Endothelin B1, (ETB1) receptor polypeptide and its encoding nucleic acid methods, and uses thereof

ABSTRACT

A new endothelin B receptor has been identified, and the amino acid and nucleotide sequence of the receptor are provided. The nucleotide sequence is useful to construct expression cassettes and vectors to produce host cells which are capable of expressing the receptor, its mutants, fragments, or fusions. Such polypeptides are useful for identifying new endothelin agonists and antagonists.

DESCRIPTION

1. Technical Field

This invention relates to the fields of molecular biology andpharmaceutical research. More specifically, this invention relates tothe identification and recombinant expression of a new endothelin Breceptor, named ETB₁, and its use to measure endothelin signaltransduction and identification of new endothelin agonists andantagonists.

2. Background of the Invention

Endothelin-1 is a 21-amino acid peptide produced by vascular endothelialcells. Endothelin-2 and endothelin-3 are closely related peptides.Endothelins have a potent vasoconstrictive effect and a sustained,potent pressor effect, which are mediated by binding of endothelins totheir receptors.

Increased endothelin levels are associated with cardiogenic shock,hypertension, pulmonary hypertension, acute myocardial infarction,uremia, Crohn's disease, ulcerative colitis, and are also observedfollowing orthotopic liver transplantation and major abdominal surgicalprocedures. Endothelin may have a pathophysiologic role in sepsis,congestive heart failure, coronary spasm, cyclosporine nephrotoxicity,vasculitis, and pregnancy-associated toxemia.

Only two endothelin receptors are presently known, ETA and ETB. The cDNAsequence of the ETB ₁ receptor is reported in Nakamuta et al.,Biochem &Biophys Res Comm 177(1): 34-39 (1991); Ogawa et al., Biochem & BiophysRes Comm 178(1): 248-255 (1991); and Sakamoto et al., Biochem & BiophysRes Comm 178(2): 656-663 (1991). The amino acid sequence of the ETAreceptor is reported in Nakamuta et al., and Ogawa et al., supra. Studyof their amino acid sequences shows that ETA and ETB are members of theseven transmembrane receptor family. This type of receptor containsseven helical domains which span the cell membrane. The seventransmembrane regions are linked by three intracellular and threeextracellular loops; in addition, the receptor possesses anextracellular amino terminal tail and an intracellular carboxyl terminaltail.

The extra- and intracellular loops contribute to the endothelin bindingand the signal transduction activity of the receptor. For example, theintracellular domains of the receptor are known to be coupled toguanyl-nucleotide-binding proteins, or G-proteins. G-proteinsinterconvert between GDP- and GTP-binding forms. Binding of endothelinto the receptor triggers the conversion of the G-protein to itsGTP-binding form, which initiates the cascade of reactions to generatethe desired biological response. This cascade is called signaltransduction. Specifically, signal transduction of the endothelinreceptors causes an increase of intracellular Ca²⁺ levels and activationof phospholipase C. Signal transduction can be measured by observing thelevels of inositol triphosphate (IP₃) and diacylglycerol (DAG), whichare increased due to phospholise C activation and cyclic AMP (cAMP).

Though the role of the G-proteins has been elucidated, the intracellularloop interactions with these proteins and with other proteins areunknown.

DISCLOSURE OF THE INVENTION

Applicants have identified a new endothelin receptor, named endothelinB₁. receptor (ETB₁ receptor). This receptor contains a decapeptideinsert in the second cytoplasmic loop which are not present in the knownendothelin B receptor. The decapeptide insert is shown in bold in SEQ IDNO: 1, amino acid numbers 199 to 208.

The decapeptide insert is encoded by last 30 nucleotides of intron 2 ofhuman ETB receptor gene. The gene thus provides a template for twodifferent RNA splice variants. The ETB ₁ receptor cDNA splice variantappears to be species specific. as shown by PCR studies and the bovineintron sequences reported in Mizuno et al., Biochem J 287: 305-309(1992). Though, in Arai et al., J Biol Chem 268(5): 3463-3470 (1993),some human intron sequences have been reported for the ETB receptorgene, not all the possible splice sites have been identified. Theputative splice sites were determined by the reported cDNA sequences.Applicants discovered that the ETB₁, mRNA is present in very lowconcentrations.

Thus, it is an object of the invention to provide native endothelin B₁receptor substantially free of nucleic acids, for identifying newendothelin agonists and antagonists.

Another object of the invention is to provide endothelin B₁ receptorpolypeptides that are mutants, fragments, and fusions of the newlyidentified endothelin B₁ receptor.

Yet another object of the invention is to provide polynucleotides thatencode the mutants, fragments, and fusions, as well as the nativeendothelin B₁ receptor. These DNA molecules can be operably linked toheterologous promoters and origins of replication to constructexpression vectors. The vectors can be introduced into suitable hostcells for endothelin B₁ receptor polypeptides expression.

Another object of the invention is to provide a host cell containing apolynucleotide molecule encoding an endothelin B₁ receptor polypeptideoperably linked to a heterologous promoter.

A further object of the invention is to provide a method for producingendothelin B₁ receptor polypeptides. The method comprises:

(a) providing a host cell comprising a vector having at least a promoteroperably linked to a DNA molecule encoding an endothelin B₁ receptorpolypeptide wherein said promoter is heterologous to said DNA molecule;and

(b) culturing the host cell under conditions which induce expression ofthe endothelin B₁ receptor polypeptide.

Yet another object of the invention is to provide method for determiningendothelin B₁ receptor signal transduction activity to identifyendothelin agonists or antagonists. The method comprises:

(a) providing a cell expressing an endothelin B₁ receptor polypeptide;

(b) exposing the expressed endothelin B1 receptor polypeptide to asubstrate; and

(c) measuring endothelin B1 receptor signal transduction activity.

Another object of the invention is to provide a method for detectingendothelin B₁ receptor polynucleotides. The method comprises:

(a) providing a nucleic acid probe which hybridizes to SEQ ID NO: 2;

(b) hybridizing a sample of polynucleotides to said probe to form aduplex; and

(c) detecting said duplexes.

MODES OF CARRYING OUT THE INVENTION A. Definitions

As used herein, the term "endothelin B₁ receptor" refers to thepolypeptides found in nature with substantial amino acid sequenceidentity to SEQ ID NO: 1 and containing the decapeptide, SLKYNSIFIF, atapproximately amino acid position 199 to 208. The decapeptide insertdistinguishes these endothelin B₁ receptors from known endothelin Breceptors.

"Endothelin B₁ receptor polypeptides" include mutants, fragments, andfusions as well as the native endothelin B₁ receptor. "Mutants" of thenative endothelin B₁ receptor are polypeptides having an amino acidsequence which retain at least 50% amino acid sequence identity with SEQID NO: 1; more typically, at least 60%; even more typically, at least80%. Preferably mutants will retain at least 85% amino acid sequenceidentity with SEQ ID NO: 1; more preferably, at least 90%; even morepreferably, at least 95%. These differences may be conservative aminoacid substitutions or deletions in the amino acid sequence. "Fragments"possess the same amino acid sequence of the native endothelin B₁receptor polypeptide except the fragments lack the amino and/or carboxylterminal sequences of the native ETB₁ I receptor, "Fusions" are mutants,fragments, or the native ETB₁ receptor that also include amino and/orcarboxyl terminal amino acid extensions. The number or type of the aminoacid substitutions is not critical, nor is the length or number of theamino acid deletions, or amino acid extensions that are incorporated inthe endothelin B₁ receptor polypeptides. However, all of thesepolypeptides will exhibit at least 20% of the native endothelin B₁receptor signal transduction activity. More typically, the polypeptidesexhibit at least 40%, even more typically the polypeptides exhibit atleast 60% of the native endothelin B, receptor signal transductionactivity. All these polypeptides will retain at least than 50% aminoacid identity with SEQ ID NO: 1; more typically, at least 60%; even moretypically, at least 80%. Preferably, these polypeptides will retain atleast 85% amino acid sequence identity with SEQ ID NO: 1; morepreferably, at least 90%; even more preferably, at least 95%.

"Signal transduction activity" occurs when binding of endothelin-1, 2,or 3, to the ETB₁ receptor triggers the desired biological response in acell or cell extract. The biological response is the result of a cascadeof biochemical reactions. Measurement of any one of these reactions canindicate that the desired biological response was triggered. Forexample, ETB₁ receptor is a G-coupled protein which, when proper signaltransduction activity occurs, triggers an increase of intracellularCa²⁺, lP₃, and DAG levels. An assay for increased levels of freecytosolic Ca²⁺ is described in Sakurai et al., EP 480 381, and Adachi etal., FEBS Lett 311(2): 179-183 (1992). Intracellular lP₃ concentrationscan be measured according to Sakurai et al., EP 480 381 and Amersham'sinositol 1,4,5-trisphosphate assay system (Arlington Heights, Ill.,U.S.A.). These assays are effective for determining ETB₁ receptor signaltransduction activity whether the receptor is normally expressed by thecell or expressed by a heterologous cell type by recombinant techniques.Proper signal transduction activity depend not only on receptor/ligandbinding but also depend on the presence of the needed intracellularproteins. Thus, though a number of cells are capable, via recombinanttechniques, of expressing ETB₁ receptor polypeptides, no biologicalresponse will be detected despite proper receptor/ligand binding if thehost cell does not produce the needed intracellular proteins. Signaltransduction activity can be detected in cells that are known to expressthe ETB₁ receptor in humans, such as heart, lung, brain, and placentalcells. Heterologous host cells, COS and Chinese Hamster Ovary (CHO)cells, for instance, can the desired biological response if altered toproduce the receptor by recombinant techniques.

A composition containing A is "substantially free of" B when at least85% by weight of the total A+B in the composition is A. Preferably, Acomprises at least about 90% by weight of the total of A+B in thecomposition, more preferably at least about 95% or even 99% by weight.

A "promoter" is a DNA sequence that initiates and regulates thetranscription of a coding sequence when the promoter is operably linkedto the coding sequence. A promoter is "heterologous" to the codingsequence when the promoter is not operably linked to the coding sequencein nature. A "native" promoter is operably linked to the coding sequencein nature.

An "origin of replication" is a DNA sequence that initiates andregulates replication of polynucleotides, such as an expression vector.The origin of replication behaves as an autonomous unit ofpolynucleotide replication within a cell, capable of replication underits own control. With certain origins of replication, an expressionvector can be reproduced at a high copy number in the presence of theappropriate proteins within the cell. Examples of origins are the 2μ andautonomously replicating sequences, which are effective in yeast; andthe viral T-antigen, effective in COS-7 cells.

Host cells capable of producing ETB₁ receptor polypeptides are cultured"under conditions inducing expression." Such conditions allowtranscription and translation of the DNA molecule encoding the ETB₁receptor polypeptide. These conditions include cultivation temperature,oxygen concentration, media composition, pH, etc. For example, if thetrp promoter is utilized in the expression vector, the media will lacktryptophan to trigger the promoter and induce expression. The exactconditions will vary from host cell to host cell and from expressionvector to expression vector.

A nucleic acid probe is said to "hybridize" with SEQ ID NO:2 if theprobe can form a duplex or double stranded complex, which is stableenough to be detected. Hybridization of the probe to a polynucleotidehaving the nucleic acid sequence of SEQ ID NO:2 depends on (1) thesequence of the nucleic acid probe and (2) the hybridization conditions.The sequence of the probe need not be exactly complementary. Forexample, a non-complementary nucleotide sequence may be attached to the5' end of the probe, with the remainder of the probe sequence beingcomplementary to SEQ ID NO:2. Alternatively, non-complementary bases orlonger sequences can be interspersed into the probe, provided that theprobe sequence has sufficient complementarity with SEQ ID NO: 2 tohybridize therewith and thereby form a duplex which can be detected. Theexact length and sequence of the probe will depend on the hybridizationconditions, such as temperature, salt condition and the like. Forexample, for diagnostic applications, depending on the complexity of theanalyte sequence, the nucleic acid probe typically contains 15-25 ormore nucleotides, although it may contain fewer nucleotides. Shortprimers generally require cooler temperatures to form sufficientlystable hybrid complexes with the template.

As used herein, the term "antibody" refers to a polypeptide or group ofpolypeptides composed of at least one antibody combining site. An"antibody combining site" is the three-dimensional binding space with aninternal surface shape and charge distribution complementary to thefeatures of an epitope of an antigen, which allows a binding of theantibody with the antigen. "Antibody" includes, for example, vertebrateantibodies, hybrid antibodies, chimeric antibodies, altered antibodies,univalent antibodies, the Fab proteins, and single domain antibodies.Antibodies do not possess the signal transduction activity of ETB₁receptor polypeptides.

B. General Method

This invention provides the amino acid and nucleotide sequence of theETB₁ receptor. With these disclosed sequences, nucleic acid probe assaysand expression cassettes and vectors for ETB₁ receptor polypeptides canbe produced. The expression vectors can be transformed into host cellsto produce ETB₁ receptor polypeptides. The purified polypeptides can beused to produce antibodies to distinguishes ETB receptors from ETB₁receptor polypeptides. Also, the host cells or extracts can be utilizedfor biological assays to isolate endothelin agonists or antagonists.

Nucleic Acid ETB₁ Receptor Probe Assays

Expression of ETB₁ receptor mRNA is maximal in, but not limited to,brain and placental cells. In contrast, ETB₁ receptor mRNA is present inheart, lung, brain, and placental cells, but is absent in uterinepoly(A⁺) RNA. This data suggests that the ETB₁ transcript is tissuespecific. This variation of mRNA levels in different cell types can beexploited with nucleic acid probe assays to determine tissue types. Forexample, PCR, branched DNA probe assays, or blotting techniquesutilizing nucleic acid probes substantially identical or complementaryto SEQ ID NO:2 can determine the presence of ETB₁ cDNA or mRNA and theabsence of ETB cDNA or mRNA.

For tissue typing, the nucleic acid probes will hybridize by thenucleotide sequence encoding the decapeptide insert of ETB₁ receptor,shown in SEQ ID NO:2 or the complement of SEQ ID NO:2. Though manydifferent nucleotide sequences will encode the decapeptide insert, SEQID NO:2 is preferred because it is the actual sequence present in humancells. Because cDNA is complementary to mRNA, for cDNA detection, thenucleic acid probe will hybridize complement of SEQ ID NO:2. Incontrast, for mRNA detection, the nucleic acid probe will hybridize toSEQ ID NO:2, itself. The nucleic acid probe sequences need not beidentical to SEQ ID NO:2 or its complement. Some variation in thesequence and length can lead to increased assay sensitivity if thenucleic acid probe can form a duplex with target nucleotides, which canbe detected. Also, the nucleic acid probe can include additionalnucleotides which bind to ETB₁ receptor sequence flanking thedecapeptide insert to stabilize the formed duplex. Or, additionalnon-ETB₁ receptor sequence may be helpful as a label to detect theformed duplex.

Probes of at least 20 nucleotides, more preferably at least 30nucleotides are useful in the nucleic acid probe assays described below.

These probes may be produced by synthetic procedures, such as thetriester method of Matteucci et al. (J. Am. Chem. Soc. (1981) 103:3185),or according to Urdea et al. Proc. Natl. Acad. Sci. USA 80: 7461 (1983),or using commercially available automated oligonucleotide synthesizers.

One example of a nucleotide hybridization assay is described in Urdea etal., PCT W092/02526 and Urdea et al., U.S. Pat. No. 5,124,246, hereinincorporated by reference. The references describe an example of asandwich nucleotide hybridization assay. The described assay utilizes amicrotiter plate as a solid support and five sets of oligonucleotides todetect the target sequences. The five oligonucleotide sets are:

(1) plate binding oligonucleotides (oligonucleotide attached to thesolid phase in Urdea et al.),

(2) capture oligonucleotides ("capture probes" in Urdea et al.),

(3) labeled probes ("amplifier probes" in Urdea et al.),

(4) branched amplifier oligonucleotides ("multimer" in Urdea et al.),and

(5) enzyme-linked oligonucleotides ("labeled oligonucleotide" in Urdeaet al.).

A microtiter plate is coated with the plate binding oligonucleotides(1). These plate binding oligonucleotides contain a sequence that iscomplementary to a sequence on the capture oligonucleotides (2). Thecapture oligonucleotides also comprise a second sequence that canhybridize to the target nucleic acids. Via the plate binding and captureoligonucleotides, the target nucleic acids are immobilized to themicrotiter plate and separated from unwanted and unbound nucleotides bysimply washing the plate.

The target nucleic acids are detected via a labeled probe (3). For thisspecific assay, the labeled probe comprises a region complementary tothe target nucleic acids and region(s) complementary to a region on thebranched amplifier oligonucleotides (4). The branched amplifieroligonucleotide comprises multiple regions, which hybridize with aregion on the enzyme-linked oligonucleotides (5). The enzyme-linkedoligonucleotides cleave light producing molecules that can be detectedwith a luminometer.

Alternatively, the Polymerase Chain Reaction (PCR) is another well-knownmeans for detecting small amounts of target nucleic acids. The assay isdescribed in Mullis et al., Meth. Enzymol. 155: 335-350 (1987); U.S.Pat. No. 4,683,195; and U.S. Pat. No. 4,683,202, incorporated herein byreference. This method, unfortunately, cannot quantitate the amount oftarget nucleic acids. Two "primer" nucleotides hybridize with the targetnucleic acids and are used to prime the reaction. The primers may becomposed of sequence within or flanking the decapeptide insert or both.The primers need not hybridize to SEQ ID NO:2 or its complement. Athermostable polymerase creates copies of target nucleic acids from theprimers using the original target nucleic acids as a template. After alarge amount of target nucleic acids are generated by the polymerase,they can be detected by more traditional methods, such as Southernblots. When using the Southern blot method, the labelled probe willhybridize to SEQ ID NO:2 or its complement.

Finally, mRNA or cDNA can be detected by traditional blotting techniquesdescribed in Sambrook et al., "Molecular Cloning: A Laboratory Manual"(New York, Cold Spring Harbor Laboratory, 1989). mRNA or cDNA generatedfrom mRNA using a polymerase enzyme can be purified and separated usinggel electrophoresis. The nucleic acids on the gel are then blotted ontoa solid support, such as nitrocellulose. The solid support is exposed toa labelled probe and then washed to remove any unhybridized probe. Next,the duplexes containing the labeled probe are detected. Typically, theprobe is labelled with radioactivity.

Expression of ETB₁ Receptor Polypeptides

Preferably, ETB₁ receptor polypeptides are produced by recombinantlyengineered host cells. These host cells are constructed by theintroduction of an expression vector composed of at least a promoteroperably linked to an ETB₁ receptor polypeptide coding sequence.

Such coding sequences can be constructed by synthesizing the entire geneor by altering an existing ETB₁ receptor polypeptide coding sequence.ETB₁ receptor polypeptides can be divided into four general categories:mutants, fragments, fusions, and the native ETB₁ receptor polypeptides.The native ETB₁ receptor polypeptides are those that occur in nature.The amino acid sequence of such polypeptides may vary slightly, lessthan by 10 amino acids from SEQ ID NO: 1, but will retain thedecapeptide insert, which distinguishes ETB₁ receptors from the knownETB receptor. The native ETB₁ receptor polypeptide coding sequence canbe selected based on the amino acid sequence shown in SEQ ID NO: 1. Forexample, synthetic genes can be made using codons preferred by the hostcell to encode the desired polypeptide. (See Urdea et al., Proc. Natl.Acad. Sci. USA 80: 7461 (1983).) Alternatively, the desired native ETB₁receptor polypeptide coding sequence can be cloned from nucleic acidlibraries using probes based on the nucleic acid sequence shown in EP480 381 or Arai et al., J Biol Chem 268(5): 3463-3470 (1993), forexample. Techniques for producing and probing nucleic acid sequencelibraries are described, for example, in Sambrook et al., "MolecularCloning: A Laboratory Manual" (New York, Cold Spring Harbor Laboratory,1989). Other recombinant techniques, such as site specific mutagenesis,PCR, enzymatic digestion and ligation, can also be used to construct thedesired ETB₁ receptor polypeptide coding sequence.

The native ETB₁ receptor polypeptide coding sequence can be easilymodified to create the other classes of ETB₁ receptor polypeptides. Forexample, mutants can be created by making conservative amino acidsubstitutions that maintain or enhance ETB₁ receptor polypeptide signaltransduction activity. The following are examples of conservativesubstitutions: Gly←→Ala; Val←→Ile←→Leu; Asp←→Glu; Lys←→Arg; Asn←→Gin;and Phe←→Trp←→Tyr. A subset of mutants, called muteins, is a group ofpolypeptides with the non-disulfide bond participating cysteinessubstituted with a neutral amino acid, generally, with serines. Thesemutants may be stable over a broader temperature range than native ETB₁receptor polypeptides. Mutants can also contain amino acid deletions orinsertions compared to the native ETB₁ receptor polypeptides. Mutantsmay include substitutions, insertions, and deletions outside thedecapeptide insert region of ETB₁ receptor. Mutants will retain at least20% of the signal transduction activity of the native ETB₁ receptorpolypeptides. The coding sequence of mutants can be constructed by invitro mutagenesis of the native ETB₁ receptor polypeptide codingsequences.

Fragments differ from mutant or native ETB₁ receptor polypeptides byamino and/or carboxyl terminal amino acid deletions. The number of aminoacids that are truncated is not critical as long as the ETB₁ receptorfragment retains at least 20% of the signal transduction activity of thenative ETB₁ receptor polypeptide. The coding sequence of such fragmentscan be easily constructed by cleaving the unwanted nucleotides from themutant or native ETB₁ receptor polypeptide coding sequences.

Fusions are fragment, mutant, or native ETB₁ receptor polypeptides withadditional amino acids at either or both of the termini. The additionalamino acid sequence is not necessarily homologous to sequence found innative ETB₁ receptor polypeptides. The fusions, just as all ETB₁receptor polypeptides, retain at least 20% of the signal transductionactivity of the native ETB₁ receptor polypeptides. Coding sequence ofthe fusions can be constructed by ligating synthetic polynucleotidesencoding the additional amino acids to fragment, mutant, or native ETB₁coding sequences. ETB₁ receptor signal transduction activity of the ETB₁receptor polypeptides can be determined by the methods described infra.

At the minimum, an expression vector will contain a promoter which isoperable in the host cell and operably linked to an ETB₁ receptorpolypeptide coding sequence. Expression vectors may also include signalsequences, terminators, selectable markers, origins of replication, andsequences homologous to host cell sequences. These additional elementsare optional but can be included to optimize expression.

A promoter is a DNA sequence upstream or 5' to the ETB₁ receptorpolypeptide coding sequence to be expressed. The promoter will initiateand regulate expression of the coding sequence in the desired host cell.To initiate expression, promoter sequences bind RNA polymerase andinitiate the downstream (3') transcription of a coding sequence (e.g.structural gene) into mRNA. A promoter may also have DNA sequences thatregulate the rate of expression by enhancing or specifically inducing orrepressing transcription. These sequences can overlap the sequences thatinitiate expression. Most host cell systems include regulatory sequenceswithin the promoter sequences. For example, when a repressor proteinbinds to the lac operon, an E. coli regulatory promoter sequence,transcription of the downstream gene is inhibited. Another example isthe yeast alcohol dehydrogenase promoter, which has an upstreamactivator sequence (UAS) that modulates expression in the absence of areadily available source of glucose. Additionally, some viral enhancersnot only amplify but also regulate expression in mammalian cells. Theseenhancers can be incorporated into mammalian promoter sequences, and thepromoter will become active only in the presence of an inducer, such asa hormone or enzyme substrate (Sassone-Corsi and Borelli (1986) TrendsGenet. 2:215; Maniatis et al. (1987) Science 236:1237).

Functional non-natural promoters may also be used, for example,synthetic promoters based on a consensus sequence of differentpromoters. Also, effective promoters can contain a regulatory regionlinked with a heterologous expression initiation region. Examples ofhybrid promoters are the E. coli lac operator linked to the E. coli tactranscription activation region; the yeast alcohol dehydrogenase (ADH)regulatory sequence linked to the yeastglyceraldehyde-3-phosphate-dehydrogenase (GAPDH) transcriptionactivation region (U.S. Pat. Nos. 4,876,197 and 4,880,734, incorporatedherein by reference); and the cytomegalovirus (CMV) enhancer linked tothe SV40 (simian virus) promoter.

A ETB₁ receptor polypeptide coding sequence may also be linked inreading frame to a signal sequence. The signal sequence fragmenttypically encodes a peptide comprised of hydrophobic amino acids whichdirects the ETB₁ receptor polypeptide to the cell membrane. Preferably,there are processing sites encoded between the leader fragment and thegene or fragment thereof that can be cleaved either in vivo or in vitro.DNA encoding suitable signal sequences can be derived from genes forsecreted endogenous host cell proteins, such as the yeast invertase gene(EP 12 873; JP 62,096,086), the A-factor gene (U.S. Pat. No. 4,588,684),interferon signal sequence (EP 60 057).

A preferred class of secretion leaders, for yeast expression, are thosethat employ a fragment of the yeast alpha-factor gene, which containsboth a "pre" signal sequence, and a "pro" region. The types ofalpha-factor fragments that can be employed include the full-lengthpre-pro alpha factor leader (about 83 amino acid residues) as well astruncated alpha-factor leaders (typically about 25 to about 50 aminoacid residues) (U.S. Pat. Nos. 4,546,083 and 4,870,008, incorporatedherein by reference; EP 324 274). Additional leaders employing analpha-factor leader fragment that provides for secretion include hybridalpha-factor leaders made with a presequence of a first yeast signalsequence, but a pro-region from a second yeast alpha-factor. (See e.g.,PCT WO 89/02463.)

Typically, terminators are regulatory sequences, such as polyadenylationand transcription termination sequences, located 3' or downstream of thestop codon of the coding sequences. Usually, the terminator of nativehost cell proteins are operable when attached 3' of the ETB₁ receptorpolypeptide coding sequences. Examples are the Saccharomyces cerevisiaealpha-factor terminator and the baculovirus terminator. Further, viralterminators are also operable in certain host cells; for instance, theSV40 terminator is functional in CHO cells.

For convenience, selectable markers, an origin of replication, andhomologous host cells sequences may optionally be included in anexpression vector. A selectable marker can be used to screen for hostcells that potentially contain the expression vector. Such markers mayrender the host cell immune to drugs such as ampicillin,chloramphenicol, erythromycin, neomycin, and tetracycline. Also, markersmay be biosynthetic genes, such as those in the histidine, tryptophan,and leucine pathways. Thus, when leucine is absent from the media, forexample, only the cells with a biosynthetic gene in the leucine pathwaywill survive.

An origin of replication may be needed for the expression vector toreplicate in the host cell. Certain origins of replication enable anexpression vector to be reproduced at a high copy number in the presenceof the appropriate proteins within the cell. Examples of origins are the2μ and autonomously replicating sequences, which are effective in yeast;and the viral T-antigen, effective in COS-7 cells.

Expression vectors may be integrated into the host cell genome or remainautonomous within the cell. Polynucleotide sequences homologous tosequences within the host cell genome may be needed to integrate theexpression cassette. The homologous sequences do not always need to belinked to the expression vector to be effective. For example, expressionvectors can integrate into the CHO genome via an unattacheddihydrofolate reductase gene. In yeast, it is more advantageous if thehomologous sequences flank the expression cassette. Particularly usefulhomologous yeast genome sequences are those disclosed in PCT W090/01800,and the HIS4 gene sequences, described in Genbank, accession no. J01331.

The choice of promoter, terminator, and other optional elements of anexpression vector will also depend on the host cell chosen. Theinvention is not dependent on the host cell selected. Convenience andthe level of protein expression will dictate the optimal host cell. Avariety of hosts for expression are known in the art and available fromthe American Type Culture Collection (ATCC). Bacterial hosts suitablefor expressing an ETB₁ receptor polypeptide include, without limitation:Campylobacter, Bacillus, Escherichia, Lactobacillus, Pseudomonas,Staphylococcus, and Streptococcus. Yeast hosts from the following generamay be utilized: Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, and Yarrowia. Immortalized mammalianhost cells include but are not limited to CHO cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and other cell lines. Anumber of insect cell hosts are also available for expression ofheterologous proteins: Aedes aegypti, Bombyx mori, Drosophilamelanogaster, and Spodoptera frugiperda (PCT WO 89/046699; Carbonell etal., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith etal., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al.(1989) In Vitro Cell. Dev. Biol. 25:225).

Transformation

After vector construction, the desired ETB₁ receptor polypeptideexpression vector is inserted into the host cell. Many transformationtechniques exist for inserting expression vectors into bacterial, yeast,insect, and mammalian cells. The transformation procedure to introducethe expression vector depends upon the host to be transformed.

Methods of introducing exogenous DNA into bacterial hosts are well-knownin the art, and typically protocol includes either treating the bacteriawith CaCl₂ or other agents, such as divalent cations and DMSO. DNA canalso be introduced into bacterial cells by electroporation or viralinfection. Transformation procedures usually vary with the bacterialspecies to be transformed. See e.g., (Masson et al. (1989) FEMSMicrobiol. Lett. 60:273; Palva et al. (1982) Proc. NatI. Acad. Sci. USA79:5582; EP Publ. Nos. 036 259 and 063 953; PCT WO 84/04541, Bacillus),(Miller et al. (1988) Proc. Nat. Acad. Sci. 85:856; Wang et al. (1990)J. Bacteriol. 172:949, Campylobacter), (Cohen et al. (1973) Proc. Natl.Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127;Kushner (1978) "An improved method for transformation of Escherichiacoli with ColE1-derived plasmids in Genetic Engineering: Proceedings ofthe International Symposium on Genetic Engineering (eds. H. W. Boyer andS. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988)Biochim. Biophys. Acta 949:318; Escherichia), (Chassy et al. (1987) FEMSMicrobiol. Lett. 44:173 Lactobacillus); (Fiedler et al. (1988) Anal.Biochem 170:38, Pseudomonas); (Augustin et al. (1990) FEMS Microbiol.Lett. 66:203, Staphylococcus), (Barany et al. (1980) J. Bacteriol.144:698; Harlander (1987) "Transformation of Streptococcus lactis byelectroporation," in Streptococcal Genetics (ed. J. Ferretti and R.Curtiss III); Perry et al. (1981) Infec. Immun. 32:1295; Powell et al.(1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4thEvr. Cong. Biotechnology 1:412, Streptococcus).

Transformation methods for yeast hosts are well-known in the art, andtypically include either the transformation of spheroplasts or of intactyeast cells treated with alkali cations. Electroporation is anothermeans for transforming yeast hosts. See for example, Methods inEnzymology, Volume 194, 1991, "Guide to Yeast Genetics and MolecularBiology." Transformation procedures usually vary with the yeast speciesto be transformed. See e.g., (Kurtz et al. (1986) Mol. Cell. Biol.6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida);(Gleeson et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al.(1986) Mol. Gen. Genet. 202:302; Hansenula); (Das et al. (1984) J.Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol.154:1165; Van den Berg et al. (1990) Bio/Technology 8:135;Kluyveromyces); (Cregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze etal. (1985) J. Basic Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 and4,929,555; Pichia); (Hinnen et al. (I1978) Proc. Natl. Acad. Sci. USA75; 1929; Ito et al. (1983) J. Bacteriol. 153:163 Saccharomyces); (Beachand Nurse (1981) Nature 300:706; Schizosaccharomyces); (Davidow et al.(1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49;Yarrowia).

Methods for introducing heterologous polynucleotides into mammaliancells are known in the art and include viral infection, dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

The method for construction of an expression vector for transformationof insect cells for expression of recombinant herein is slightlydifferent than that generally applicable to the construction of abacterial expression vector, a yeast expression vector, or a mammalianexpression vector. In an embodiment of the present invention, abaculovirus vector is constructed in accordance with techniques that areknown in the art, for example, as described in Kitts et al.,BioTechniques 14: 810-817 (1993), Smith et al., Mol. Cell. Biol. 3: 2156(1983), and Luckow and Summer, Virol. 17: 31 (1989). In one embodimentof the present invention, a baculovirus expression vector is constructedsubstantially in accordance to Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987). Moreover, materials andmethods for baculovirus/insect cell expression systems are commerciallyavailable in kit form, for example, the MaxBac® kit from Invitrogen (SanDiego, Calif.).

Also, methods for introducing heterologous DNA into an insect host cellare known in the art. For example, an insect cell can be infected with avirus containing an ETB₁ receptor polypeptide coding sequence. When thevirus is replicating in the infected cell, the ETB₁ receptor polypeptidewill be expressed if operably linked to a suitable promoter. A varietyof suitable insect cells and viruses are known and include followingwithout limitation.

Insect cells from any order of the Class Insecta can be grown in themedia of this invention. The orders Diptera and Lepidoptera arepreferred. Example of insect species are listed in Weiss et al., "CellCulture Methods for Large-Scale Propagation of Baculoviruses," inGranados et al. (eds.), The Biology of Baculoviruses: Vol. II PracticalApplication for Insect Control, pp. 63-87 at p. 64 (1987). Insect celllines derived from the following insects are exemplary: Carpocapsapomeonella (preferably, cell line CP-128); Trichoplusia ni (preferably,cell line TN-368); Autograph californica; Spodoptera frugiperda(preferably, cell line Sf9); Lymantria dispar; Mamestra brassicae; Aedesalbopictus; Orgyia pseudotsugata; Neodiprio sertifer; Aedes aegypti;Antheraea eucalypti; Gnorimoschema operceullela; Galleria mellonella;Spodoptera littolaris; Blatella germanic; Drosophila melanogaster;Heliothis zea; Spodoptera exigua; Rachiplusia ou; Plodia interpunctella;Amsaeta moorei; Agrotis c-nigrum, Adoxophyes orana; Agrotis segetum;Bombyx mori; Hyponomeuta malinellu;, Colias eurytheme; Anticarsiagermmetalia; Apanteles melanoscelu; Arctia caja; and Porthetria dispar.Preferred insect cell lines are from Spodoptera frugiperda, andespecially preferred is cell line Sf9. The Sf9 cell line used in theexamples herein was obtained from Max D. Summers (Texas A & MUniversity, College Station, Texas, 77843, U.S.A.) Other S. frugiperdacell lines, such as IPL-Sf-21AE III, are described in Vaughn et al., InVitro 13: 213-217 (1977).

The insect cell lines of this invention are suitable for thereproduction of numerous insect-pathogenic viruses such as parvoviruses,pox viruses, baculoviruses and rhabdcoviruses, of whichnucleopolyhedrosis viruses (NPV) and granulosis viruses (GV) from thegroup of baculoviruses are preferred. Further preferred are NPV virusessuch as those from Autographa spp., Spodoptera spp., Trichoplusia spp.,Rachiplusia spp., Gallerai spp., and Lymantria spp. More prefferred arebaculovirus strain Autographa californica NPV (AcNPV), Rachiplusia ouNPV, Galleria mellonella NPV, and any plaque purified strains of AcNPV,such as E2, R9, S1, M3, characterized and described by Smith et al., JVirol 30: 828-838 (1979); Smith et al., J Virol 33: 311-319 (1980); andSmith et al., Virol 89: 517-527 (1978).

Typically, insect cells Spodoptera frugiperda type 9 (SF9) are infectedwith baculovirus strain Autographa californica NPV (AcNPV) containing anETB₁ receptor polypeptide coding sequence. Such a baculovirus isproduced by homologous recombination between a transfer vectorcontaining the coding sequence and baculovirus sequences and a genomicbaculovirus DNA. Preferably, the genomic baculovirus DNA is linearizedand contains a disfunctional essential gene. The transfer vector,preferably, contains the nucleotide sequences needed to restore thedisfunctional gene and a baculovirus polyhedrin promoter and terminatoroperably linked to the ETB₁ receptor polypeptide coding sequence. (SeeKitts et al., BioTechniques 14(5): 810-817 (1993).

The transfer vector and linearized baculovirus genome are transfectedinto SF9 insect cells, and the resulting viruses probably containing thedesired coding sequence. Without a functional essential gene thebaculovirus genome cannot produce a viable virus. Thus, the viableviruses from the transfection most likely contain the ETB₁ receptorpolypeptide coding sequence and the needed essential gene sequences fromthe transfer vector. Further, lack of occlusion bodies in the infectedcells are another verification that the ETB₁ receptor polypeptide codingsequence was incorporated into the baculovirus genome.

The essential gene and the polyhedrin gene flank each other in thebaculovirus genome. The coding sequence in the transfer vector isflanked at its 5' with the essential gene sequences and the polyhedrinpromoter and at its 3' with the polyhedrin terminator. Thus, when thedesired recombination event occurs the ETB₁ receptor polypeptide codingsequence displaces the baculovirus polyhedrin gene. Such baculoviruseswithout a polyhedrin gene will not produce occlusion bodies in theinfected cells. Of course, another means for determining if codingsequence was incorporated into the baculovirus genome is to sequence therecombinant baculovirus genomic DNA. Alternatively, expression of thedesired ETB₁ receptor polypeptide by cells infected with the recombinantbaculovirus is another verification means.

Monitoring ETB₁ Receptor Polypeptide Expression Levels

Immunoassays and ligand binding assays can be utilized to determine ifthe transformed host cell is expressing the desired ETB₁ receptorpolypeptide.

For example, an immunofluorescence assay can be easily performed ontransformed host cells without separating the ETB₁ receptor polypeptidesfrom the cell membrane. The host cells are first fixed onto a solidsupport, such as a microscope slide or microtiter well. This fixing steppermeabilizes the cell membrane. Next, the fixed host cells are exposedto an anti-ETB₁ receptor polypeptide antibody. Preferably, to increasethe sensitivity of the assay, the fixed cells are exposed to a secondantibody, which is labelled and binds to the anti-ETB₁ receptorpolypeptide antibody. Typically, the secondary antibody is labelled withan fluorescent marker. The host cells which express the ETB₁ receptorpolypeptides will be fluorescently labelled and easily visualized underthe microscope. See, for example, Hashido et al., Biochem & Biophys ResComm 187(3): 1241-1248 (1992).

Also, the ETB₁ receptor polypeptides do not need to be separated fromthe cell membrane for ligand binding assay. The host cells may be fixedto a solid support, such as a microtiter plate. Alternatively, a crudemembrane fraction can be separated from lysed host cells bycentrifugation (See Adachi et al., FEBS Lett 311(2): 179-183 (1992)).The fixed host cells or the crude membrane fraction is exposed tolabelled endothelin, or other suitable ligand such as an endothelinagonist or antagonist. Typically, the endothelin is labelled withradioactive atoms. The host cells which express the desired ETB₁receptor polypeptide will bind with the labelled ligand which can beeasily detected.

Purification

The purified ETB₁ receptor polypeptides are useful for signaltransduction assays, ligand/receptor binding assays. The purifiedpolypeptides can also be utilized to produce ETB₁ receptor polypeptidespecific antibodies.

For ligand/receptor binding studies, the crude cell membrane fractionscan be utilized. These membrane extracts can be isolated from cellswhich expressed ETB₁ receptor polypeptides by lysing the cells andseparating the cell membrane fraction from the intracellular fractionsby centrifugation. See Adachi et al., FEBS Lett 311 (2): 179-183 (1992)for an endothelin binding assay procedure using cell membranes.Alternatively, whole cells, expressing ETB₁ receptor polypeptides, canbe cultured in a microtiter plate, for example, and used for endothelinbinding assay. See Sakamoto et al., Biochem & Biophys Res Comm 178(2):656-663 (1991) for a description of such an assay.

Once the polypeptide has been dissociated from the cell membrane, thedesired ETB₁ receptor polypeptide can also be affinity purified withspecific ETB₁ antibodies.

Antibodies

Antibodies against ETB₁ receptor polypeptides are useful for affinitychromatography, immunofluorescent assays, and distinguishing ETB fromETB₁ receptor polypeptides. Antibodies which recognize the decapeptideinsert are of particular interest.

Such antibodies can be used to distinguish ETB₁ receptor polypeptidesfrom ETB receptors. These antibodies are useful in immunofluorescentassays when the cells are processed so that the membrane is madepermeable. The permeablization of the cell membrane permits theantibodies to bind to the decapeptide insert of the second cytoplasmicloop of the ETB₁ receptor polypeptides. Peptides containing thedecapeptide insert, SLKYNSIFIF, can be easily synthesized using knownautomated synthesizer and gel purified for antibody production.

Antibodies to the proteins of the invention, both polyclonal andmonoclonal, may be prepared by conventional methods. In general, theprotein is first used to immunize a suitable animal, preferably a mouse,rat, rabbit or goat. Rabbits and goats are preferred for the preparationof polyclonal sera due to the volume of serum obtainable, and theavailability of labeled anti-rabbit and anti-goat antibodies.Immunization is generally performed by mixing or emulsifying the proteinin saline, preferably in an adjuvant such as Freund's complete adjuvant,and injecting the mixture or emulsion parenterally (generallysubcutaneously or intramuscularly). A dose of 50-200 μg/injection istypically sufficient. Immunization is generally boosted 2-6 weeks laterwith one or more injections of the protein in saline, preferably usingFreund's incomplete adjuvant. One may alternatively generate antibodiesby in vitro immunization using methods known in the art, which for thepurposes of this invention is considered equivalent to in vivoimmunization.

Polyclonal antisera is obtained by bleeding the immunized animal into aglass or plastic container, incubating the blood at 25° C. for one hour,followed by incubating at 4° C. for 2-18 hours. The serum is recoveredby centrifugation (e.g., 1,000 ×g for 10 minutes). About 20-50 ml perbleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the method of Kohler andMilstein, Nature (1975) 256:495-96, or a modification thereof.Typically, a mouse or rat is immunized as described above. However,rather than bleeding the animal to extract serum, the spleen (andoptionally several large lymph nodes) is removed and dissociated intosingle cells. If desired, the spleen cells may be screened (afterremoval of nonspecifically adherent cells) by applying a cell suspensionto a plate or well coated with the protein antigen. B-cells expressingmembrane-bound immunoglobulin specific for the antigen bind to theplate, and are not rinsed away with the rest of the suspension.Resulting B-cells, or all dissociated spleen cells, are then induced tofuse with myeloma cells to form hybridomas, and are cultured in aselective medium (e.g., hypoxanthine, aminopterin, thymidine medium,"HAT"). The resulting hybridomas are plated by limiting dilution, andare assayed for the production of antibodies which bind specifically tothe immunizing antigen (and which do not bind to unrelated antigens).The selected MAb-secreting hybridomas are then cultured either in vitro(e.g., in tissue culture bottles or hollow fiber reactors), or in vivo(as ascites in mice).

If desired, the antibodies (whether polyclonal or monoclonal) may belabeled using conventional techniques. Suitable labels includefluorophores, chromophores, radioactive atoms (particularly ³² P and ¹²⁵I), electron-dense reagents, enzymes, and ligands having specificbinding partners. Enzymes are typically detected by their activity. Forexample, horseradish peroxidase is usually detected by its ability toconvert 3,3', 5,5'-tetramethylbenzidine (TNB) to a blue pigment,quantifiable with a spectrophotometer. "Specific binding partner" refersto a protein capable of binding a ligand molecule with high specificity,as for example in the case of an antigen and a monoclonal antibodyspecific therefor. Other specific binding partners include biotin andavidin or streptavidin, IgG and protein A, and the numerousreceptor-ligand couples known in the art. It should be understood thatthe above description is not meant to categorize the various labels intodistinct classes, as the same label may serve in several differentmodes. For example, ¹²⁵ I may serve as a radioactive label or as anelectron-dense reagent. HRP may serve as enzyme or as antigen for a MAb.Further, one may combine various labels for desired effect. For example,MAbs and avidin also require labels in the practice of this invention:thus, one might label a MAb with biotin, and detect its presence withavidin labeled with ¹²⁵ I, or with an anti-biotin MAb labeled with HRP.Other permutations and possibilities will be readily apparent to thoseof ordinary skill in the art, and are considered as equivalents withinthe scope of the instant invention.

Use in Biological Assays

ETB₁ receptor polypeptides can also be used to screen peptide librariesto determine the amino acid sequence of endothelin peptide agonist orantagonists.

A "library" of peptides may be synthesized following the methodsdisclosed in U.S. Pat. No. 5,010,175, and in PCT W091/17823, bothincorporated herein by reference in full. Briefly, one prepares amixture of peptides, which is then screened to determine the peptidesexhibiting the desired signal transduction and receptor bindingactivity. In the '175 method, a suitable peptide synthesis support(e.g., a resin) is coupled to a mixture of appropriately protected,activated amino acids. The concentration of each amino acid in thereaction mixture is balanced or adjusted in inverse proportion to itscoupling reaction rate so that the product is an equimolar mixture ofamino acids coupled to the starting resin. The bound amino acids arethen deprotected, and reacted with another balanced amino acid mixtureto form an equimolar mixture of all possible dipeptides. This process isrepeated until a mixture of peptides of the desired length (e.g.,hexamers) is formed. Note that one need not include all amino acids ineach step: one may include only one or two amino acids in some steps(e.g., where it is known that a particular amino acid is essential in agiven position), thus reducing the complexity of the mixture. After thesynthesis of the peptide library is completed, the mixture of peptidesis screened for binding to the selected ETB₁ receptor polypeptide. Thepeptides are then tested for their ability to inhibit or enhance ETB₁receptor signal transduction activity. Peptides exhibiting the desiredactivity are then isolated and sequenced.

The method described in '17823 is similar. However, instead of reactingthe synthesis resin with a mixture of activated amino acids, the resinis divided into twenty equal portions (or into a number of portionscorresponding to the number of different amino acids to be added in thatstep), and each amino acid is coupled individually to its portion ofresin. The resin portions are then combined, mixed, and again dividedinto a number of equal portions for reaction with the second amino acid.In this manner, each reaction may be easily driven to completion.Additionally, one may maintain separate "subpools" by treating portionsin parallel, rather than combining all resins at each step. Thissimplifies the process of determining which peptides are responsible forany observed receptor binding or signal transduction activity.

In such cases, the subpools containing, e.g., 1-2,000 candidates eachare exposed to the desired ETB₁ receptor polypeptide. Each subpool thatproduces a positive result is then resynthesized as a group of smallersubpools (sub-subpools) containing, e.g., 20-100 candidates, andreassayed. Positive sub-subpools may be resynthesized as individualcompounds, and assayed finally to determine the peptides, which exhibita high binding constant. Then, these peptides can be tested for theirability to inhibit or enhance the ETB₁ signal transduction activity. Themethods described in '17823 and U.S. Pat. No. 5,194,392 (hereinincorporated by reference) enable the preparation of such pools andsubpools by automated techniques in parallel, such that all synthesisand resynthesis may be performed in a matter of days.

Endothelin peptide agonists or antagonists are screened using anyavailable method. The methods described herein are presently preferred.The assay conditions ideally should resemble the conditions under whichthe ETB₁ receptor signal transduction is exhibited in vivo, i.e., underphysiologic pH, temperature, ionic strength, etc. Suitable agonists orantagonists will exhibit strong inhibition or enhancement of the ETB₁,signal transduction activity at concentrations which do not raise toxicside effects in the subject. Agonists or antagonists which compete forbinding to the ETB₁ receptor ligand binding site may requireconcentrations equal to or greater than the ETB₁ receptor concentration,while inhibitors capable of binding irreversibly to the ETB₁ receptormay be added in concentrations on the order of the ETB₁ receptorconcentration.

Signal Transduction Assays

Most cellular Ca²⁺ ions are sequestered in the mitochondria, endoplasmicreticulum, and other cytoplasmic vesicles, but binding of endothelin toETB₁ will trigger the increase of free Ca²⁺ ions in the cytoplasm. Withfluorescent dyes, such as fura-2, the concentration of free Ca²⁺ can bemonitored. The ester of fura-2 is added to the media of the host cellsexpressing ETB₁ receptor polypeptides. The ester of fura-2 is lipophilicand diffuses across the membrane. Once inside the cell, the fura-2 esteris hydrolyzed by cytosolic esterases to its non-lipophilic form, andthen the dye cannot diffuse back out of the cell. The non-lipophilicform of fura-2 will fluoresce when it binds to the free Ca²⁺ ions, whichare released after binding of a ligand to the ETB₁ receptor. Thefluorescence can be measured without lysing the cells at an excitationspectrum of 340 nm or 380 nm and at fluorescence spectrum of 500 nm. SeeSakurai et al., EP 480 381 and Adachi et al., FEBS Lett 311(2): 179-183(1992) for examples of assays measuring free intracellular Ca²⁺concentrations.

The rise of free cytosolic Ca²⁺ concentrations is preceded by thehydrolysis of phosphatidylinositol 4,5-bisphosphate. Hydrolysis of thisphospholipid by the plasmamembrane enzyme phospholipase C yields1,2-diacylglycerol (DAG), which remains in the membrane, and thewater-soluble inosital 1,4,5-trisphosphate (IP₃). Binding of endothelinor endothelin agonists will increase the concentration of DAG and IP₃.Thus, signal transduction activity can be measured by monitoring theconcentration of these hydrolysis products.

To measure the IP₃ concentrations, radioactively labelled ³ H-inositolis added to the media of host cells expressing ETB₁ receptorpolypeptides. The ³ H-inositol taken up by the cells and afterstimulation of the cells with endothelin or endothelin agonist, theresulting inositol triphosphate is separated from the mono anddi-phosphate forms and measured. See Sakurai et al., EP 480 381.Alternatively, Amersham provides an inosital 1,4,5-trisphosphate assaysystem. With this system Amersham provides tritylated inosital1,4,5-trisphosphate and a receptor capable of distinguishing theradioactive inositol from other inositol phosphates. With these reagentsan effective and accurate competition assay can be performed todetermine the inositol triphosphate levels.

C. EXAMPLES

The examples presented below are provided as a further guide to thepractitioner of ordinary skill in the art, and are not to be construedas limiting the invention in any way.

EXAMPLE 1 Tissue Typing by PCR Utilizing ETB₁ Insert Specific Primers

Tissue distribution of the mRNA encoding the native ETB₁ receptor wasperformed by reverse transcriptase PCR.

A. RNA Isolation from Tissue

Approximately 0.1 g of tissue is minced in ice or homogenized at roomtemperature with 1 ml of denaturing solution (4M guanidiniumthiocyanate; 25 mM sodium citrate, pH 7.0; 0.5% (wt/v) sarcosyl; 0.1Mbeta mercaptoethanol). Sequentially, 0.1 ml of 2M sodium acetate, pH 4.0is added, then 1 ml phenol, and finally 0.2 ml chloroform/isoamylalcohol (49:1) are added to the homogenate, and the sample is cooled onice for 15 minutes. Samples are centrifuged at 10,000 g for twentyminutes at 4° C. The aqueous phase, containing the RNA, is removed fromthe pellet. Next, the aqueous phase is mixed with 1.0 ml isopropanol andincubated at -20° C. for one hour to precipitate the RNA. The RNA ispelleted from the sample by centrifugation at 10,000 g for 20 minutes,and the resulting pellet was dissolved in 0.3 ml of denaturing solution.The redissolved pellet is transferred into a 1.5 ml Eppendorf tube, andthe RNA is precipitated with one volume of isopropanol and incubated at-20° C. for one hour. After centrifugation in an Eppendorf centrifugefor ten minutes at 4° C., the aqueous phase is removed, and the RNApellet is washed in 75% (v/v) ethanol. The RNA pellet is dissolved in 50μl TE (10 mM Tris, pH 7.0 and 1 mM EDTA).

B. Generating cDNA by Reverse Transcriptase Polymerase Chain Reaction(RTPCR) from RNA

cDNA can be synthesized from RNA isolated from the desired tissue, orRNA from human tissues can be purchased from Clontech, Palo Alto,Calif., U.S.A.

cDNA was synthesized from 5 μg of human brain poly (A+) RNA, fromClontech, Palo Alto, Calif., U.S.A., using a 3' RACE (RapidAmplification of cDNA Ends) kit from Life Technologies, Gaithersburg,Md., U.S.A. This procedure was designed to convert up to 1 μg of totalRNA into first strand cDNA. Although poly(A)⁺ RNA can be used in thisprotocol, this level of purity is typically not necessary. A control RNAwas included in the kit to aid in verification of that the reactionperforms correctly. To use the control RNA as a template for the cDNAsynthesis, 2 μl of the control RNA (100 ng) can be substituted in thecDNA reaction in place of the sample RNA. (Note: Each component wasmixed and quickly centrifuged before each use.)

Five μg of total or poly (A)⁺ selected RNA was added to 13 μl ofDEPC-treated water to a 0.5 ml microcentrifuge tube. Next, 1 μl of the10 μM solution containing primer Vet119 was added to the tube and mixedgently. The sequence, 5' to 3' of Vet119 is AC TAG TAC TTT TTT TTT TTTTTT TTT (SEQ ID NO:3). The mixture was heated to 65° C. for 10 minutesand chilled for two minutes on ice. The contents of the tube werecentrifuged and the following was added:

10 X synthesis buffer . . . 2 μl

10 mM dNTP mix . . . 1 μl

0.1 M DTT . . . 2 μl

The final composition of the mixture was

20 mM Tris-HC1 (pH 8.4);

50 mM KCl;

2.5 mM MgCl₂ ;

100 μg/ml bovine serum albumin (BSA);

10 mM DTT;

500 nM Vet 119;

500 μM each of dATP, dCTP, dGTP, dTTP; and

≦1 μg (<50 ng/,μl) of RNA.

The mixture was mixed gently and spun down by brief centrifugation. Themixture was equilibrated to 42° C. for 2 minutes. Next, 1 μl ofSUPERSCRIPT RT was added. The mixture was then incubated in a 42° C.water bath or heat block for 30 minutes. The mixture was brieflycentrifuged to collect the liquid at the bottom of the tube. The mixturewas then placed on ice and 1 μl of RNase H was added to the tube andmixed. The mixture was incubated at 42° C. for 10 minutes.

The resulting cDNA was purified by adsorption to GLASSMAX filters fromLife Technologies, Gaithersburg, Md., U.S.A. Ninety five μl of bindingbuffer was added to the cDNA reaction mixture. The entire mixture wastransferred to a GLASSMAX Spin Cartridge. The cartridge was centrifugedat 13,000 g for 20 seconds. The flowthrough material was retained in aseparate tube. Next, the cartridge was washed with 400 μl of cold 1Xwash buffer, and the cartridge was centrifuged at 13,000 g for 20seconds, and the flowthrough material was discarded. This wash step wasrepeated two more times. Finally, the cartridge was washed with 400 μlof cold 70% ethanol. The cartridge was centrifuged at 13,000 g for 20seconds, and the flowthrough material was discarded. Next, the cartridgewas centrifuged for 1 minute at 13,000 g; then, the cartridge wasinserted into a new collection tube, and 50 μl distilled water at 65° C.was added. The cartridge was centrifuged at 13,000 g for 20 seconds. Theflowthrough material contained the cDNA material for amplification.

C. Tissue Typing by PCR

Two μl of the eluted material was used as a template for PCR. One pmoleof each primer and 1 unit of polymerase from Perkin Elmer, Norwalk,Conn., U.S.A., were added to a final volume of 50 μl, and included 2.5μl formamide, 10 mM-Tris/HCI (pH 8.3), 50 mM-KCl, 3.5 mM-MgCl₂, and 0.2mM dNTPs. Amplification was accomplished by performing PCR for 40 cyclesat 94° C. for 1 minute, 50° C. for 1 minute, and 72° C. for 2 minutes.Five μl of the product were electrophoresed to visualize PCR products.All PCR products were cloned using the TA cloning kit (from Invitrogen,San Diego, Calif., U.S.A.) and sequenced by cycle sequencing using anApplied Biosystems sequencer according to established techniques. Allsequences were confirmed by sequencing both strands.

Oligo dT primed cDNA was amplified with insert specific primer Vet71,SEQ ID NO:4, and ETB specific primer Vet85, SEQ ID NO:5 The sequence ofthe primers was as follows:

Vet71: 5' CAG CTT AAA ATA CAA TTC TAT TTT TAT CTT 3'

Vet85: 5' GGR ARC CAG CAR AGR GCA AA 3'.

PCR products were obtained with human brain, heart, lung, and placenta,but no PCR products were obtained from uterus poly(A+) RNA. In a controlreaction with ETB specific primers, Vet100, SEQ ID NO:22, and Vet85, SEQID NO:4, all samples yielded PCR products. The sequence of Vet100 was 5'CTG TGC TGA GTC TAT GTG CT 3'.

EXAMPLE 2 Construction of a ETB₁ Coding Sequence

The coding sequence for ETB receptor was isolated from poly(A)⁺ RNA. DNAencoding the decapeptide sequence of SEQ ID NO:2 was incorporated byoverlap PCR. The final ETB₁, coding sequence encodes the amino acidsequence of SEQ ID NO: 1. The coding sequence contains XmaI and XbaIends for cloning convenience.

The coding sequence was constructed from the cDNA of the ETB codingsequence synthesized from isolated poly(A⁺) RNA from human brain. cDNAwas synthesized from the RNA according to Example 1, section B. The RNAwas purchased from Clontech, Palo Alto, Calif., U.S.A.

The ETB receptor polypeptide cDNA was isolated from the poly(A⁺) RNA byPCR. Two μl of the RNA was used as template for the reaction with thefollowing reagents: 1 pmole of primers Vet126 (SEQ ID NO:6) and Vetl27(SEQ ID NO:7), 1 unit of polymerase from Perkin Elmer, Norwalk, Conn.,U.S.A., 2.5 μl formamide, 10 mM-Tris/HCI (pH 8.3), 50 mM-KCI, 3.5mM-MgCl₂, 0.2 mM dNTPs and water was added to a final volume of 50 μl.The PCR product was cloned into PCR-II vector from Invitrogen, SanDiego, Calif., U.S.A. The sequence of the primers,(SEQ ID NO:5) and (SEQID NO:) was as follows:

Vetl26: 5' ATA CCC GGG ACC ATG CAG CCG CCT CCA AGT C 3'

Vetl27: 5' ATA TCT AGA TCA AGA TGA GCT GTA TTT AT 3'

The native ETB₁ receptor polypeptide coding sequence was constructed byoverlap PCR according to Shyamala et al., Gene 97: 1-6 (1991). This PCRtechnique permits the incorporation of mutations within a desired DNAfragment, such as a complete coding sequence. The desired DNA fragmentis first synthesized in two double stranded parts. The DNA sequence ofthe first part is the 5' end of the desired fragment to the mutagenesissite, and the DNA sequence second part contains the remainder of thedesired DNA fragment from the site of mutagenesis to the 3' end. Theseparts are constructed using PCR with the mutation incorporated into thePCR primers. Thus, two sets of primers are utilized.

The desired primers are kinased, so that the PCR products which resultfrom the elongation of these primers can be preferentially digested byan exonuclease. The result of this digestion is two single strandedproducts that overlap and can hybridize at the mutatgenesis site.Together, the single stranded products span the entire sequence of thedesired fragment. Then PCR is used to create a complete double strandedDNA fragment. This technique was used to incorporate the decapeptideinsert into the native ETB receptor polypeptide coding sequence.

Primers Vet 122, SEQ ID NO:23 and Vet 123, SEQ ID NO:24, were kinased asdescribed below. The sequence of the primers was as follows:

Vet122: 5' TAC AAT TCT ATT TTT ATC TTC AGA TAT CGA GCT GTT GC 3'

Vet123: 5' AAA AAT AGA ATT GTA TTT TAA GCT GTC AAT ACT CAG AGC 3'

The primers were added to a solution whose final volume was 13 μl. Thesolution consisted of 1 μl of 0.2 M ATP, 10 μl of 50 μM primers, 1.3 μlof 10X kinasing buffer, and 1 μl polynucleotide kinase from New EnglandBiolabs, Boston, Mass., U.S.A.,. The solution was incubated at 37° C.for 15 minutes.

Two μl of PCR-II vector with the native ETB₁ receptor polypeptide codingsequence was used as a template for the PCR. Two PCR reactionscontaining the following reagents were performed sequentially: 2.5 μlformamide, 10 mM Tris/HCI (pH 8.3), 50 mM KCI, 3.5 mM MgCI₂, 0.2 mMdNTPs, as described in section B, and 1 pmole of each of the desiredprimers, the final reaction volume was 50 μl. One reaction was with Vet122 and M13 forward primers, and the second reaction was with Vet 123and M13 reverse primers. The sequence of the M13 forward and reverseprimers were as follows:

M13 Forward: 5' AGG GTT TTC CCA GTC ACG AC 3' (SEQ ID NO:8)

M13 Reverse: 5' GAT AAC AAT TTC ACA CAG GA 3' (SEQ ID NO:9)

Thirty repetitions of the following temperature cycle was used: 94° C.for 1 minute, 50° C. for 1 minute, and 72° C. for 2 minutes.

Next, the double stranded products were digested with λ exonuclease toproduce the desired single stranded products. After both PCR reactions,5 μl of 67 mM glycine, titrated to pH 9.4 with NaOH, was added to 40 μlof the final amplification reaction. Then, 5 units of λ exonuclease(BRL, Gaithersburg, Md., U.S.A.) was added to the solution and incubatedat 37° C. for 30 minutes. The digest DNA was passed through a SephacrylS-300 spin column.

To facilitate overlap hybridization, 15 μl of the pass through from thetwo reaction were mixed with the amplification reagents as describedabove and extended with the addition of Klenow at each cycle for 5temperature cycles of 94° C. for 1 minute denaturation; and 37° C. for 1minute extension. After hybridization, PCR techniques were used tocreate the desired double stranded ETB₁ receptor polypeptide codingsequence. One unit of Taq polymerase was added to the solutioncontaining the hybridized fragments, and the solution was subjected to20 temperature cycles of 94° C. for 1 minute, 50° C. for 1 minute, and72° C. for 2 minutes. The amplified DNA was digested with XmaI and XbaIand cloned into BacPak9 and pCMV vectors.

EXAMPLE 3 Construction of a Mammalian Cell ETB₁ Receptor PolypeptideExpression Vector

The resulting XmaI/XbaI ETB₁ receptor polypeptide fragment was ligatedwith a XmaI/XbaI fragment pCMV9D, a mammalian expression vector. pCMV9Dcontains the cytomegalovirs immediate early 1 enhancer/promoter, a polyA sequence terminator, the simian virus 40 origin of replication, theampicillin resistance gene, and mouse dihydrofolate reductase (DHFR)gene under the control of the adenovirus promoter. pCMV9D is aderivative of the pCMV6a expression vector, which has been altered toinclude the DHFR selectable marker.

Plasmid pCMV6a is a mammalian cell expression vector which contains thetranscriptional regulatory region from human cytomegalovirus majorimmediate early region (HCMV IE1) of the Towne strain. The plasmidcontains the SV40 polyadenylation region derived from pSV7d (describedin U.S. Pat. No. 5,156,949) as a 700 bp PvuI-Sall fragment; the SV40origin of replication derived from pSVT2 (Meyers et al., Cell 25:373-384 (1981); Rio et al., Cell 32: 1227-1240 (1983)) as a 1.4 kbPvuI-EcoRl (the ends were filled in with Klenow); and the HCMV IEIpromoter as a 1.7 kb SspI-Sall fragment derived from a subclone of thehuman cytomegalovirus (Towne Strain). The HCMV IE1 promoter regioncontains the region encoding the first exon (5' untranslated), the firstintron and the start of the second exon (where the Sall site was createdby in vitro mutagenesis). This intron is included on the assumption thatspliced transcripts lead to faster processing and more stable mRNA. Theexpression vector also has an SV40 origin of replication to permittransient expression in COS7 cells, and a bacterial β-lactamase gene topermit DNA cloning by selection for ampicillin resistance.

The DHFR selectable marker was isolated from pAdDHFR. This vectorincludes the adenovirus-2 major late promoter (Ad-MLP, map units16-27.3) fused to the 5' untranslated sequences of the mouse DHFR cDNA(Nunberg et al., Cell 19: 355-364 (1980)). A fragment from pAdDHFRcontaining the promoter and DHFR cDNA was inserted into pCMV6a to createpCMV9D.

EXAMPLE 4 Construction of a COS Cell Line Expressing ETB₁ ReceptorPolypeptide

The mammalian cell expression vector, VCE40, is introduced into COS-7,monkey kidney cells by electroporation. The electroporated cellstransiently expressed the native ETB₁ receptor polypeptide.

A monolayer of confluent COS-7 cells were rinsed once with calcium andmagnesium free phosphate buffered saline (CMF-PBS). Five ml oftrypsin/EDTA solution was added to the culture media to detach the cellsfrom the solid support. The cells were incubated in the trypsin/EDTAsolution at 37° C. until the cells detached. The composition of thetrypsin/EDTA solution was: 1.6 g KCI, 32 g NaCI, 4 g glucose, 2.32 gsodium bicarbonate, 10 g trypsin, 0.4 g EDTA, 0.016 g Phenol red in afinal volume of 4 L of water titrated to pH 7.0-7.1 with HCI.

Next, the cells were resuspended into a tube containing 5 mls 10% (v/v)fetal calf serum in DME with glutamine (from the University ofCalifornia, San Francisco), 1.0M HEPES, to inhibit the trypsin enzymaticactivity. The detached cells were centrifuged in a table-top centrifugeat 1,000 g for 3 minutes and then resuspended in PBS. This step wasrepeated twice. After the final centrifugation, the cells areresuspended at a concentration of 1×10⁷ cells/ml in 10 mM HEPES/PBS. Twohundred and fifty μl of the cell suspension was added to theelectroporation cuvette with 0.4 cm electrode gaps from BIORAD(Hercules, Calif., U.S.A.) with 10 μg of pCM9DETB₁.

The cells were electroporated in a BIORAD gene pulser apparatus, catalogno. 165-2076 with a capacitance extender, also from BIORAD (Hercules,Calif., U.S.A.), catalog no. 165-2087. The apparatus was set at 400Volts and 250/μFd. The cells were pulsed at room temperature. Afterelectroporation, the cells were vigorously pipetted to disperse theclumps of cells. Approximately, 1 ml of 10% (v/v) FCS in DME was addedto the cuvette, and this cell suspension was plated onto a 100 mm platewith 10 ml of 10% (v/v) FCS in DME.

Expression of the native ETB₁ receptor polypeptide is determined byWestern utilizing rabbit polyclonal antibodies which bind specificallyto the following amino acid sequences (1) EEKQSCLKFKANDHG (SEQ ID NO:10), (2) PRTISPPPCQGPIEIC (SEQ ID NO: 11), (3) DPNRCELLSFLL (SEQ ID NO:12), and (4) SLKYNSIFIFC (SEQ ID NO: 13). Expression can also beconfirmed by ligand binding assays, as described in Example 5.

After transfection, the cells are washed with PBS and released from theculture plates by scraping the cells into PBS containing 5 mM EDTA and aprotease cocktail. The protease cocktail contains 0.5 mM PMSF, 5 μg/mlaprotinin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin. Cells are harvestedby centrifugation at 2,000 ×g for 4 minutes at 4° C. and resuspeded in 1ml PBS-protease cocktail. The harvested cells are lysed by rapidlydiluting the cells into 20 ml of ice-cold 20 mM HEPES buffer, pH 7.5containing protease cocktail. The lysed cells are centrifuged at 30,000g for 30 minutes. The pellet, containing the cell membranes, isseparated from the aqueous phase, and then the pellet is resuspended in50 mM HEPES, pH 7.5, 10 mM MgCI₂.

The cell membrane fraction is run on a 10% acrylamide SDS-PAGE gel. Thegel is blotted overnight onto Immobilon-P (from Millipore, Bedford,Mass., U.S.A.) at 25 volts. The filter is blocked with 5% milk powder,0.5 M NaCI, 20 mM Tris, pH 7.5, and 0. 1% Tween® 20 for 1 hour at roomtemperature.

The IgG fraction of the first antibody is diluted 1:100 (˜1 μg/ml) intoblocking solution. The filter is incubated in this solution for 1 hourat room temperature. Next, the filter is washed three times with 0.5 MNaCI, 20 mM Tris, pH 7.5, and 0. 1% Tween 20. The filter is incubated inthe solution for ten minutes for each wash.

The second antibody, a goat anti-rabbit horse conjugated to horse radishperoxidase (Boehringer Mannheim, Cincinnati, Ohio, U.S.A.), is diluted1:30,000 in blocking solution. The filter is incubated in this solutionfor 1 hour at room temperature. Next, the filter is washed three timeswith 0.5 M NaCl, 20 mM Tris, pH 7.5, and 0.1% Tween 20. The filter isincubated in the solution for ten minutes for each wash.

The bound antibodies are detected by ECL (enhanced chemiluminescence)using detection reagents by Amersham.

EXAMPLE 5 Construction of a CHO Cell Line Expressing ETB₁ ReceptorPolypeptide

One of the most commonly used methods of introducing DNA into mammaliancells is to coprecipitate the DNA with calcium phosphate and present themixture to cells. The specific procedure used to transfect ChineseHamster Ovary (CHO) cells with the plasmid VCE 40 is as follows.

DG44 CHO cells were seeded at 7.1×10⁵ cells per 60 mm dish in 10% fetalbovine serum (FBS) in Ham's F12. The cells were grown about 24 hoursuntil the dishes were 80% confluent. On the day of the transfection thefollowing DNA-calcium phosphate coprecipitate was prepared:

    ______________________________________                                               VCE40          40.5 μl                                                     TE.sup.1       399.5 μl                                                    2x HBS.sup.2   500 μl                                                      2 M CaCl.sub.2.sup.3                                                                         60 μl                                                ______________________________________                                         .sup.1 TE = 10 mM TrisHCl, 1 mM EDTA pH 8.0                                   .sup.2 HBS = Hanks' balanced salts: 1.4 mM Na.sub.2 HPO.sub.4, 10 mM KCl,     12 mM glucose, 275 mM NaCl, and 40 mM HEPES, pH 6.95.                         .sup.3 2 M CaCl.sub.2 is diluted in 10 mM HEPES, pH 5.8.                 

The solution was mixed and incubated at room temperature for 30 minutes.Then, the medium was removed from the cells and 9 ml of fresh media wasadded to the dishes. The DNA-calcium phosphate coprecipitate was mixedby pipetting the solution and 1 ml of the DNA coprecipitate was added toeach dish.

The cells were incubated with the DNA coprecipitate at 37° C. for 4hours. The medium was removed from the cells and replaced with 2 ml of20% (w/v) dimethyl sulfoxide (DMSO) in 1×HBS. After 2 minute incubationat room Temperature, 4 ml of serum-free Ham's F12 medium was added. Themedium is removed and the cells were washed twice with 4 ml ofserum-free Ham's F12 medium. Finally, 4 ml of 10% FBS in Ham's F12medium was added, and the cells were incubated at 37° C. for 24 hours.

The medium was removed and 4 ml of the selective medium was added. Theselective medium was composed of 10% dialyzed FBS in Ham's F12 (withouthypoxanthine and thymidine), 10,000 U/L penicillin, 10 mg/Lstreptomycin, and 2.5 mg/L amphotericin B. The clones were picked andscreened using a ligand binding assay.

The cells from the clone were plated with 10 ml medium in 100 mm plates.When the plates are ˜90% confluent, the media is removed and the platesare rinsed with 5.0 ml of calcium and magnesium free PBS with 6 mM EDTA.Next, 5.0 ml of the PBS/EDTA medium is added to the plates and themedium is pipetted up and down to dislodge the cells from the plate. Themedium with the cells is saved for assay. The plates are washed with 2.5ml of the PBS/EDTA media and is added to the 5.0 ml of dislodged cells.Ten ml of binding buffer is added to the cells to dilute out the EDTA.The composition of the binding buffer is 25 mM HEPES, 150 mM NaCl, 5 mMCaCl₂, 1 mg/ml bovine serum albumin (BSA). The cells are centrifuged topellet the cells and remove the supernatant. The cells are resuspendedin 5.0 ml of binding buffer 100 μl of this solution containing cells andradioactive endothelin was added to 300 μl of binding buffer containing0.1 nM of radioactive endothelin (either 1 or 3) from Amersham (ArlintonHeights, Ill., U.S.A). and varying concentrations of unlabeledendothelin from Bachem (Torrence, Calif., U.S.A). The recommended finalconcentrations of the unlabeled endothelin is 100 nM, 10 nM, 1 nM, and100 pM. The sample is placed on a 0.24 cm glass fiber filter fromMillipore and dried on a Millipore 1225 Sampling Manifold (cat. no.XX27-02550, Bedford, Mass., U.S.A.)

EXAMPLE 6 Construction of an Insect Cell ETB₁ Receptor PolypeptideExpression Vector

The resulting XmaI/XbaI ETB₁ receptor polypeptide fragment from Example2 was ligated with a EcoRV/XbaI fragment of ETB₁ receptor with theinsect cell expression vector, pBacPAK9, from Clontech, Palo Alto,Calif., U.S.A. pBacPAK9 contains a polylinker so that the ETB₁ receptorcoding sequence can be inserted after the polyhedrin promoter bydigesting the pBacPAK9 with XmaI and EcoRV.

The resulting insect cell expression vector was named VCE 39. Thisvector was transformed into E. coli DH5α cells and deposited with theAmerican Type Culture Collection (ATCC), Rockville, Md., U.S.A. Thetransformed cells were assigned accession no. 69321.

This expression vector is used as a transfer vector to construct abaculovirus containing the ETB₁ receptor polypeptide coding sequence. Adescription of such a vector is described below.

EXAMPLE 7 Construction of an Insect Cell Line Expressing ETB₁ ReceptorPolypeptide

1.) The Production of a Recombinant Baculovirus

The transfer vector with the KGF₁₆₃ coding sequence and its signalpeptide insert is transfected together with a mutant baculovirus intoSpodoptera frugiperda, SF9 cells. The mutant baculovirus is a derivativeof AcNPV that lacks a functional essential gene. This mutant baculovirusmust recombine with the transfer vector to produce a viable baculovirus.To increase the number of recombinants, the mutant baculovirus waslinearized with BsuI. In this regard, several BsuI sites wereincorporated into the mutant baculovirus for this purpose. This mutantbaculovirus is similar to the baculovirus described in Kitts et al.,Biotechniques 14(5): 810-817 (1993).

A.) Preparation of Recombinant Baculovirus

First, 1×10⁶ SF9 cells are seeded per well in a 6-well plate containing2 ml of a complete TNMFH medium. The cells are incubated for at least 30minutes at room temperature to allow the cells to attach to the plate.The complete TNMFH medium contained GRACE'S medium obtained from JRHBiosciences, Lenexa, Kans., U.S.A. and is supplemented with 10% (v/v)fetal bovine serum (56° C. heat inactivated for 30 minutes), 3% (w/v)Yeastolate (Difco, Detroit, Mich., U.S.A.), and I% (v/v) Fungi-BACT(Irvine Scientific, Santa Ana, Calif., U.S.A.).

For addition to each of the above wells, the following transfectionmixture is initially separately prepared by first, adding 0.5 ml ofGRACE'S medium containing no supplement into a sterile 1.5 ml eppendorftube; next, adding 0.5 μg of linearized mutant baculovirus DNA and,approximately, 2-3 μg of the transfer vector containing the KGF₁₆₃coding sequence and the signal peptide insert. This is approximately a4:1 ratio of transfer vector to mutant baculovirus. Finally, thecationic liposome solution, BRL catalog #8282SA (from BRL, Gaithersburg,Md., U.S.A.) is mixed thoroughly, and 10 μl of this liposome solutionwere added to the baculovirus mixture. This transfection mixture isincubated at room temperature for 15 minutes.

Before the transfection mixture is added to the cells in the wells, theTNMFH medium is removed therefrom, and the cells are washed with 1-2 mlof GRACE'S medium without supplements. When the transfection mixture isfinally prepared, as described above, all the medium is removed from theSF9 cells, and the transfection mixture is added dropwise to the cells.The 6-well plate is then covered with parafilm to reduce evaporation ofthe transfection mixture. The plate is rocked slowly at room temperaturefor approximately four hours on a Bellco®, catalog no. #774020020,Vineland, N.J., U.S.A., side/side rocking platform at setting 2.5.

After the incubation of the cells with the transfection mixture, 0.5 mlof complete TNMFH medium is added to the cells and the mixture isincubated at 27° C. in a humidified chamber (92%) for 48 hours.Thereafter, the medium containing recombinant baculovirus is removedfrom the cells and stored at 4° C. until the plaque assay forrecombinant virus isolation is performed. This medium constitutes theprimary source of the recombinant virus.

After the medium is removed from the cells, 2 ml of complete TNMFH isadded to the cells, and the cells are further incubated in a humidifiedchamber (92%) at 27° C. for another 48-72 hours. This final step isperformed to provide a back-up source of recombinant virus and toprovide a visual record of the viral infection.

B.) Plaque Purification of the Recombinant Baculovirus

The recombinant KGF baculoviruses prepared as above were plaque-purifiedaccording to the following steps:

First, 4 ml of SF9 cells at 5×105 cells/ml in GRACE'S medium is platedon 60 mm LUX dishes (catalog #5220). The cells are incubated at roomtemperature for 20-30 minutes to allow the cells to adhere to the plate.In the meantime, the medium containing the primary source of recombinantvirus is diluted into 2 ml of TNMFH medium at 1:10, 1:50, 1:100, and1:200 dilutions. After the cells are allowed to adhere, the medium isaspirated from the adherent cells, and the various dilutions of therecombinant virus are added quickly so as not to allow the cells to dry.The cells are then incubated for 1 hour at 27° C. in a humidifiedchamber (92%).

Ten minutes before the incubation is complete, an agarose solution isprepared. A 2× concentration of GRACE'S medium supplemented as before isheated to 37° C. Only the amount used for the assay is heated;otherwise, proteins may precipitate upon repeated heating. When the 2×medium was warm, a 3% (w/v) Sea Plaque agarose mixture in water ismelted in a microwave and immediately mixed 1:1 with the 2× medium. Theagarose solution is then allowed to cool at room temperature for severalminutes.

The viral medium is aspirated from the cells in the dishes by tiltingthe dishes slightly on the hood rim. The dishes are then drained for afew seconds and aspirated again to remove as much liquid as possible.This second aspiration step is included to reduce the likelihood ofvirus spreading and causing indistinct plaques. Six dishes or fewer arehandled at a time to avoid drying the cells. The aspirated dishes arenever left exposed with the lids off for more than a few seconds.

Next, 4 ml of the agarose solution are added to each dish and leftundisturbed for 15 minutes. The agarose overlay is dried by lifting thelids to the side of the plate to permit the agarose to dry forapproximately 25 minutes. Then, the dishes are covered with the lids,and the cells are incubated in a humidified chamber (92%) for 4 days at27° C.

To facilitate visualization of the plaques, the dishes are stained with2 ml per dish of 25% (w/v) Sea Plaque agarose in complete TNMFH mediumwith 0.01% (v/v) neutral red, from Sigma, St. Louis, Mo., U.S.A. Theagarose overlay is dried at room temperature for about one hour with thelids on, before the dishes are returned to a humidified chamber (92%) at27° C. for 3-4 hours. The neutral red dye is incorporated only by theviable cells.

When the plaques are well-resolved, 7 individual plugs are picked with asterile Pasteur pipet and each is transferred to 1 ml of complete TNMFH.The plugs are incubated for 2 days at room temperature. The plugs werethen vortexed, and the plaque assay was repeated with 50 μl of thissolution.

C.) Expansion of the Plague Purified Baculovirus

To expand the viral titers, 3-4 plugs of each baculovirus clone from thesecond round of plaque purification are placed directly onto cultures ofSf9 cells. The cells are plated at 2.5×10⁵ /well in 6-well plates with2.5 ml of complete TNMFH medium. The cells and virus were then incubatedfor approximately 4 hours at 27° C. in a humidified chamber (92%).

For the second round of expansion, all the virus from the 6-well platesare transferred to 10 cm dishes. The 10 cm dishes are plated with7.5×10⁶ cells in 7.5 ml of complete TNMFH media. The cells and virus areincubated for 48-72 hours at 27° C. in a humidified chamber (92%).

After this infection, the cells are thoroughly screened for any wildtype virus contamination that appeared as infected cells containingocclusion bodies.

2.) Insect Cell Expression of an ETB₁ Receptor Polypeptide CodingSequence

SF9 insect cells are infected with the recombinant baculovirus, asdescribed in sections A-C, to provide a large amount of recombinantnative ETB₁ receptor polypeptide for purification and analysis.

More specifically, SF9 cells are diluted to a cell density of 1×10⁶cells/ml in ISFM-7. The cells are seeded into either a 1 L or 2 L shakeflask with 300 ml or 500 ml of medium, respectively. Virus from thesecond expansion (described in section C) was used to inoculate thecells at 10% (v/v) dilution. The cells and virus are shaken atapproximately 131 rpm with the caps of the shake flasks are loosened toprovide an adequate air supply. The cells are incubated at 27° C. for 48hrs.

EXAMPLE 8 Identification of Endothelin Agonists and Antagonists

Construction of an phage library encoding random peptides is describedin Devlin, W091/18980. Such a construction consists of

(1) Producing Oligonucleotides Encoding Random Peptides;

(2) Creating a Shuttle Vector, Plasmid M13LP67, for Recombination withthe Wild Type Phage; and

(3) Production of Phage Encoding Random Peptides by Recombination.

Once the phage library is constructed, the library is screened using aETB₁ receptor polypeptide. From the phage library, peptides with thedesired binding properties can be assayed for their endothelin agonistor antagonist properties.

I. Producing Oligonucleotides Encoding Random Peptides

An oligonucleotide having the following structure was synthesized, andpurified using methods known in the art, as described in Devlin,W091/18980: 5' CTTTCTATTCTCACTCCGCTGAA(NNS)₁₅ CCGCCTCCACCTCCACC 3' (SEQID NO:14); and 5'GGC CGG TGG AGG TGG AGG CGG (iii)₁₅ TTC AGC GGA GTG AGAATA GAA AGG - TAC 3' (SEQ ID NO: 15).

During the synthesis of (NNS)₁₅, a mixture consisting of equal amountsof the deoxynucleotides A, C and T, and about 30% more G was used for N,and an equal mixture of C and G for S. Deoxyinosine (i) was used becauseof its capacity to base pair with each of the four bases (A, G, C, andT) (J.F. Reidhaar-Olson et al., Science, (1988) 24:53). Alternatively,other base analogs may be used as described by J. Habener et al., ProcNatl Acad Sci USA (1988) 85:1735.

Immediately preceding the nucleotide sequence that encodes the randompeptide sequence is a nucleotide sequence that encodes alanine andglutamic acid residues. These amino acids were included because theycorrespond to the first two amino terminal residues of the wild typemature gene III protein of M13, and thus may facilitate producing thefusion protein produced as described below.

Immediately following the random peptide sequence is a nucleotidesequence that encodes 6 proline residues. Thus, the oligonucleotideencodes the following amino acid sequence:

H₂ N-Ala-Glu-Xaal₁₅ -Pro₆ (SEQ ID NO:27)

Xaa denotes amino acids encoded by the random DNA sequence. As describedbelow, the oligonucleotides were cloned into a derivative of M13 toproduce a mature fusion protein having the above amino acid sequence,and following the proline residues, the entire wild type mature geneIII.

II. Construction the Shuttle Vector Plasmid M13LP67, for Recombinationwith the Wild Type Phage

The plasmid M13LP67 was used to express the random peptide/gene IIIfusion protein construct. M13LP67 was derived from M13 mpl9.

Briefly, Ml3mpl9 was altered in two ways. The first alteration consistedof inserting the marker gene, β-lactamase, into the polylinker region ofthe virion. This consisted of obtaining the gene by PCR amplificationfrom the plasmid pAc5. The oligonucleotide primers that were annealed tothe pAc5 template hare the following sequence:

5' GCT GCC CGA GAG ATC TGT ATA TAT GAG TAA ACT TGG 3' (SEQ ID NO:16)

5' GCA GGC TCG GGA ATT CGG GAA ATG TGC GCG GAA CCC 3' (SEQ ID NO: 17)

Amplified copies of the β-lactamase gene were digested with therestriction enzymes BglII and EcoRl, and the replicative form of themodified M13 mp19 was digested with BamHI and EcoRl. The desiredfragments were purified by gel electrophoresis, ligated, and transformedinto E. coli strain DH5 alpha (BRL). E. coli transformed with phage thatcarried the insert were selected on ampicillin plates. The phage soproduced were termed JD32.

The plasmid form of the phage, pJD32 (M13mp19Amp'), was mutagenized sothat two restriction sites, EagI and KpnI, were introduced into gene IIIwithout altering the amino acids encoded in this region. The restrictionsites were introduced using standard PCR in vitro mutagenesis techniquesas described by M. Innis et al. in "PCR Protocols--A Guide to Methodsand Applications" (1990), Academic Press, Inc.

The KpnI site was constructed by converting the sequence, TGTTCC, atposition 1611 to GGTACC. The two oligonucleotides used to effect themutagenesis have the following sequence:

LP159: AAACTTCCTCATGAAAAAGTC (SEQ ID NO:18)

LP162: AGAATAGAAAGGTACCACTAAAGGA (SEQ ID NO: 19).

To construct the EagI restriction site, the sequence at position 1631 ofpJD32, CCGCTG, was changed to CGGCCG using the following twooligonucleotides:

LP160: TTT AGT GGT ACC TTT CTA TTC TCA CTC GGC CGA AAC

TGT (SEQ ID NO:25)

LP161: AAA GCG CAG TCT CTG AAT TTA CCG (SEQ ID NO:26)

More specifically, the PCR products obtained using the primers LP159,LP162 and LP160 and LP161 were digested with BspHI and KpnI, and KpnIand AlwNI, respectively. These were ligated with T4 ligase to M13mpl9previously cut with BspHI and AlwNI to yield M13mpLP66. This vectorcontains the desired EagI and KpnI restriction sites, but lacks theampicillin resistance gene, β-lactamase. Thus, the vector M13mpLP67,which contains the EagI and KpnI restriction sites and β-lactamase wasproduced by removing the β-lactamase sequences from pJD32 by digestingthe vector with XbaI and EcoRI. The β-lactamase gene was then insertedinto the polylinker region of M13mpLP66 which was previously digestedwith XbaI and EcoRI. Subsequent ligation with T4 ligase producedM13mpLP67, which was used to generate the random peptide library.Schematics of the construction of M13mpLP67 are shown in Devlin et al.,PCT W091/18980.

Production of Phage Encoding Random Peptides

To produce phage having DNA sequences that encode random peptidesequences, M13LP67 was digested with EagI and KpnI, and ligated to theoligonucleotides produced as described in Example 1 above. The ligationmixture consisted of digested M13LP67 DNA at 45 ng/μL, a 5-fold molarexcess of oligonucleotides, 3.6 U/μL of T4 ligase (New England Biolabs),25 mM Tris-HCI, pH 7.8, 10 mM MgC'₂, 2 mM DTT, 0.4 mM ATP, and 0.1 mg/mlBSA. Prior to being added to the ligation mixture, the individualoligonucleotides were combined and heated to 95° C. for 5 minutes, andsubsequently cooled to room temperature in 15 μL aliquots. Next, theligation mixture was incubated for 4 hours at room temperature andsubsequently overnight at 15° C. This mixture was then electroporatedinto E. coli as described below.

M13LP67 DNA was electroporated into H249 cells prepared essentially asdescribed by W. Dower et al., Nuc Acids Res (1988) 16:6127. H249 cellsare a recA, sup°, F' kan^(R) derivative of MM294. Briefly, 4×10⁹ H249cells and 1 μg of M13LP67 DNA were combined in 85 μL of a lowconductivity solution consisting of 1 mM HEPES. The cell/Ml3LP67DNAmixture was positioned in a chilled 0.56 mm gap electrode of a BTXelectroporation device (BTX Corp.) and subjected to a 5 millisecondpulse of 560 volts.

Immediately following electroporation, the cells were removed from theelectrode assembly, mixed with fresh H249 lawn cells, and plated at adensity of about 2 ×10⁵ plaques per 400 cm² plate. The next day phagefrom each plate were eluted with 30 ml of fresh media, PEG precipitated,resuspended in 20% glycerol, and stored frozen at -70° C. About 2.8×10⁷plaques were harvested and several hundred analyzed to determine theapproximate number that harbor random peptide sequences. Using thepolymerase chain reaction to amplify DNA in the region that encodes therandom peptide sequence, it was determined that about 50-90% of thephage contained a 69 base pair insert at the 5' end of gene III. Thisconfirmed the presence of the oligonucleotides that encode the randompeptides sequences. The PCR reaction was conducted using standardtechniques and with the following oligonucleotides:

5' TCGAAAGCAAGCTGATAAACCG 3' (SEQ ID NO:20)

5' ACAGACAGCCCTCATAGTTAGCG 3' (SEQ ID NO:21)

The reaction was run for 40 cycles, after which the products wereresolved by electrophoresis in a 2% agarose gel. Based on these results,it was calculated that phage from the 2.8×10⁷ plaques encode about 2×10⁷different random amino acid sequences.

Panning for Endothelin Agonists and Antagonists

Peptides having an affinity for ETB₁ receptor are identified as follows:

1.) 15mer phage (2.5×10¹⁰) prepared as described above are selected bycoincubation with 10⁶ Sf9 cells expressing native ETB₁, (See Example 7)on the second day after infection. The coincubation is at roomtemperature for 60 minutes in Grace's medium with 2% nonfat milk.Binding phage are eluted with 6M urea (pH 2.2), the pH neutralized byadding 2M Tris-HCI, and assayed. The phage are amplified on solid agarplates as plaques, eluted with Tris-buffered saline, and precipitatedwith polyethylene glycol.

2.) The phage resulting from round 1 are reselected on CHO cellsexpressing the native ETB₁ (See Example 5) on second day 2 after platingthe cells at a density of 7.1 ×10⁵, using 3.1×10¹¹ phage on in DMEM with2% nonfat milk and 10 mM HEPES. The phage are bound, eluted, assayed,and amplified as described in round 1.

3.) The phage selected in round 2 are reselected on Sf9 cells expressingthe native ETB₁ receptor on day 2 post-infection as described for round1 (2.8×10¹⁰ phage on 10⁶ Sf9 cells). Sample phage from the urea eluateare cloned, and their DNAs are isolated and sequenced.

Once the amino acid sequence of the putative agonists and antagonists isdetermined, synthetic oligopeptides can be produced and their signaltransduction activity can be assayed by, for example, Amersham'sinositol 1,4,5-trisphosphate assay system (Arlington Heights, Ill.,U.S.A.).

CHO cells expressing the ETB₁ receptor polypeptide (Example 5) areplated at a density of 1×10⁵ cells/well in a 12-well plate. The cellsare cultured for 2 days, and then, the cells are washed twice with PBScontaining 0.2% BSA. Next, the cells are incubated in the same mediumfor 30 minutes at 37° C. The medium on the cells is changed to PBScontaining 0.2% BSA and 10 mM LiCl, and the cells are incubated foranother 30 minutes at 37° C.

Signal transduction is induced by changing the medium of the cells toPBS containing 0.2% BSA, 10 mM LiCl, and the desired concentration ofthe oligopeptide, as determined by the screening. The cells areincubated in this medium for 5 minutes and then the media is removedfrom the cells. Next, 0.2 volumes ice-cold 20% (v/v) perchloric acid(PCA) is added to the cells to quench the stimulation and to prepare thecells for the inositol phosphate assay. The cells are incubated on icein PCA for 20 minutes. At the beginning of the incubation, the cells aredislodged from the plate with a rubber policeman. After the incubation,the cells are removed from the plate and centrifuged at 2,000×g for 15minutes at 4° C. The supernatants are removed and titrated to pH 7.5with 10 N KOH and kept on ice. The solution is centrifuged at 2,000×gfor 15 minutes at 4° C. to remove the precipitate. The supernatant isthen assayed to determine the amount of inositol trisphosphate present.

Amersham provides a kit containing the reagents for an inositoltriphosphate competition assay. With the kit, an inositol1,4,5-trisphosphate binding protein is provided, which cross-reacts withinositol 1,3,4,5-tetrakisphophate less than 10% and less than 1% withother inositol phosphates. The assay measures that amount of inositoltriphosphate that competes for the binding protein with the radioactivelabeled triphosphate.

1.) Preparing the Standard

First all the reagents to thaw at 2-8° C. and then mix thoroughly. Asthis is occurring, label 8 poypropylene tubes (12×75 mm) "0.19," "0.38,""0.76," "1.5," "3.1," 6.2, "12.5," and "25 pmol." Pipette 1.5 ml ofwater into the tube marked "25 pmol." Into the remaining marked standardtubes pipette 500 μl of water. Next, into the marked "25 pmol" tube, addexactly 100 μl of the standard solution (3 nmol D-myo-inositol1,4,5-trisphosphate in water). Mix the solution completely. Transfer 500μl of the 25 pmol solution to the 12.5 pmol tube, and vortex thesolution throughly. Repeat this 1:2 dilution succesively with theremaining tubes. These working standards should be prepared immediatelybefore each assay and not re-used. The standard solution from the kitshould be recapped after use and immediately stored at -15° C. to -30°C.

2.) Assay Protocol

First label duplicate polypropylene tubes (10×55 mm) "TC" for totalcounts; "NSB" for non-specific binding; "B₀ " for zero standards;"0.19," "0.38," "0.76," "1.5," "3.1," 6.2 "12.5 " and "25 " pmol for thestandards; and whatever is desired for the samples. Next, pipette intoall the tubes 100 μl of the assay buffer (0.1M Tris buffer pH 9.0, 4 mMEDTA and 4 mg/mIl bovine serum albumin (BSA)). Into the B_(O) and TCtubes, 100 μl and 200 μl, respectively deionized water is added. Then,starting with the most dilute solution, pipette 100 μl of each of thestandard solutions, described above, into the appropriately labelledtubes. Use a new pipette tip for each standard solution. Add into theNSB tubes, 100 μl of stock standard solution (3 nmol D-myo-inositol1,4,5-trisphosphate in water). One hundred microliters of the samplesshould be added to the appriopriate sample tubes. Use a new pipette tipfor each sample.

One hundred microliters of first the tracer (˜1.0 μCi or ˜37kBq ofD-nyo- ³ H!inositol 1,4,5-trisphosphate in 1:1 (v/v) water:ethanol) andthen binding protein is added to all the tubes. All the tubes arevortexed to mix all the contents throughly and then incubated for 15minutes on ice. Then, the binding protein is isolated by thecentrifugation procedure below.

All the tubes, except those labelled "TC", are centrifuged at 2,000×gfor at least 10 minutes at 4° C. After centrifugation, the tubes arecarefully placed into a suitable decantation rack and the supernatant ispoured off and discarded. The tubes are kept inverted and placed onabsorbent tissues and allowed to drain for 2 minutes. Next, the rims ofthe inverted tubes are firmly blotted on the tissue to remove anyadhering droplets of liquid, and the inside of the tubes are carefullyswapped for the same reason. This is done carefully to avoid disturbingthe pellet at the bottom of the tube.

To each tube, 200 μt of water is added to resuspend the pellet exceptthe "TC" labelled tubes. The tube is vortexed to mix the solutionthroughly. Then, 2 ml of scintillation fluid is added to the resuspendedpellet. Before measuring the radioactivity of each sample for fourminutes in a 0-scintillation counter, the samples are capped and mixedthroughly.

EXAMPLE 9 Purification of ETB₁ Receptor Polypeptides from Nucleic Acids

Membrane preparation for endothelin ligand binding assay using COS-7 orCHO cells transfected with VCE 40. See Examples 4 and 5. COS-7 cells,for example, are grown in 245 mm×245 mm tissue culture plates andtransfected according to the protocol in Example 4 with 30 μg of VCE 40plasmid DNA. After two days, the cells are washed with PBS and releasedfrom the culture plates by scraping the cells into PBS containing 5 mMEDTA and a protease cocktail. The protease cocktail contains 0.5 mMPMSF, 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin. Cellsare harvested by centrifugation 2,500×g for 5 minutes at 4° C., andresuspended in 1 ml of the PBS-protease cocktail. The harvested cellsare lysed by rapidly diluting the cells into 20 ml of ice-cold 20 mMHEPES buffer, pH 7.5 containing protease cocktail. The lysed cells arecentrifuged at 30,000 g for 30 minutes. The pellet, containing the cellmembranes, is separated from the aqueous phase, and then the pellet isresuspended in 50 mM HEPES, pH 7.5, 10 mM MgCl₂. The membranes can befrozen at -70° C. for future use.

Deposit Information:

The following materials were deposited with the American Type CultureCollection:

    ______________________________________                                        Name              Deposit Date                                                                            Accession No.                                     ______________________________________                                        Escherichia coli DH5α, VCE 39                                                             4 June 1993                                                                             69321                                             Escherichia coli DH5α, VCE 40                                                             4 June 1993                                                                             69322                                             Phage Library 7.1 in M13LP67                                                                              40828                                             ______________________________________                                    

The above materials have been deposited with the American Type CultureCollection, Rockville, Md., under the accession numbers indicated. Thisdeposit will be maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for purposesof Patent Procedure. The deposits will be maintained for a period of 30years following issuance of this patent, or for the enforceable life ofthe patent, whichever is greater. Upon issuance of the patent, thedeposits will be available to the public from the ATCC withoutrestriction.

These deposits are provided merely as convenience to those of skill inthe art, and are not an admission that a deposit is required under 35U.S.C. §112. The sequence of the polynucleotides contained within thedeposited materials, as well as the amino acid sequence of thepolypeptides encoded thereby, are incorporated herein by reference andare controlling in the event of any conflict with the writtendescription of sequences herein. A license may be required to make, use,or sell the deposited materials, and no such license is granted hereby.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 27                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 452 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: -                                                               (B) LOCATION: 199..208                                                        (D) OTHER INFORMATION: /note= "Decapeptide Insert"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetGlnProProProSerLeuCysGlyArgAlaLeuValAlaLeuVal                              151015                                                                        LeuAlaCysGlyLeuSerArgIleTrpGlyGluGluArgGlyPhePro                              202530                                                                        ProAspArgAlaThrProLeuLeuGlnThrAlaGluIleMetThrPro                              354045                                                                        ProThrLysThrLeuTrpProLysGlySerAsnAlaSerLeuAlaArg                              505560                                                                        SerLeuAlaProAlaGluValProLysGlyAspArgThrAlaGlySer                              65707580                                                                      ProProArgThrIleSerProProProCysGlnGlyProIleGluIle                              859095                                                                        LysGluThrPheLysTyrIleAsnThrValValSerCysLeuValPhe                              100105110                                                                     ValLeuGlyIleIleGlyAsnSerThrLeuLeuArgIleIleTyrLys                              115120125                                                                     AsnLysCysMetArgAsnGlyProAsnIleLeuIleAlaSerLeuAla                              130135140                                                                     LeuGlyAspLeuLeuHisIleValIleAspIleProIleAsnValTyr                              145150155160                                                                  LysLeuLeuAlaGluAspTrpProPheGlyAlaGluMetCysLysLeu                              165170175                                                                     ValProPheIleGlnLysAlaSerValGlyIleThrValLeuSerLeu                              180185190                                                                     CysAlaLeuSerIleAspSerLeuLysTyrAsnSerIlePheIlePhe                              195200205                                                                     ArgTyrArgAlaValAlaSerTrpSerArgIleLysGlyIleGlyVal                              210215220                                                                     ProLysTrpThrAlaValGluIleValLeuIleTrpValValSerVal                              225230235240                                                                  ValLeuAlaValProGluAlaIleGlyPheAspIleIleThrMetAsp                              245250255                                                                     TyrLysGlySerTyrLeuArgIleCysLeuLeuHisProValGlnLys                              260265270                                                                     ThrAlaPheMetGlnPheTyrLysThrAlaLysAspTrpTrpLeuPhe                              275280285                                                                     SerPheTyrPheCysLeuProLeuAlaIleThrAlaPhePheTyrThr                              290295300                                                                     LeuMetThrCysGluMetLeuArgLysLysSerGlyMetGlnIleAla                              305310315320                                                                  LeuAsnAspHisLeuLysGlnArgArgGluValAlaLysThrValPhe                              325330335                                                                     CysLeuValLeuValPheAlaLeuCysTrpLeuProLeuHisLeuSer                              340345350                                                                     ArgIleLeuLysLeuThrLeuTyrAsnGlnAsnAspProAsnArgCys                              355360365                                                                     GluLeuLeuSerPheLeuLeuValLeuAspTyrIleGlyIleAsnMet                              370375380                                                                     AlaSerLeuAsnSerCysIleAsnProIleAlaLeuTyrLeuValSer                              385390395400                                                                  LysArgPheLysAsnCysPheLysSerCysLeuCysCysTrpCysGln                              405410415                                                                     SerPheGluGluLysGlnSerLeuGluGluLysGlnSerCysLeuLys                              420425430                                                                     PheLysAlaAsnAspHisGlyTyrAspAsnPheArgSerSerAsnLys                              435440445                                                                     TyrSerSerSer                                                                  450                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTTAAAATACAATTCTATTTTTATCTTCAG30                                              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ACTAGTACTTTTTTTTTTTTTTTTTT26                                                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CAGCTTAAAATACAATTCTATTTTTATCTT30                                              (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GGRARCCAGCARAGRGCAAA20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ATACCCGGGACCATGCAGCCGCCTCCAAGTC31                                             (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATATCTAGATCAAGATGAGCTGTATTTAT29                                               (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AGGGTTTTCCCAGTCACGAC20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GATAACAATTTCACACAGGA20                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GluGluLysGlnSerCysLeuLysPheLysAlaAsnAspHisGly                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ProArgThrIleSerProProProCysGlnGlyProIleGluIleCys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AspProAsnArgCysGluLeuLeuSerPheLeuLeu                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      SerLeuLysTyrAsnSerIlePheIlePheCys                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 85 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CTTTCTATTCTCACTCCGCTGAANNSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNSN60                NSNNSNNSCCGCCTCCACCTCCACC85                                                   (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: unsure                                                          (B) LOCATION:                                                                 (C) OTHER INFORMATION: /note= "N is inosine"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GGCCGGTGGAGGTGGAGGCGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN60                NNNNNNTTCAGCGGAGTGAGAATAGAAAGGTAC93                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GCTGCCCGAGAGATCTGTATATATGAGTAAACTTGG36                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GCAGGCTCGGGAATTCGGGAAATGTGCGCGGAACCC36                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AAACTTCCTCATGAAAAAGTC21                                                       (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AGAATAGAAAGGTACCACTAAAGGA25                                                   (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TCGAAAGCAAGCTGATAAACCG22                                                      (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ACAGACAGCCCTCATAGTTAGCG23                                                     (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CTGTGCTGAGTCTATGTGCT20                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      TACAATTCTATTTTTATCTTCAGATATCGAGCTGTTGC38                                      (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AAAAATAGAATTGTATTTTAAGCTGTCAATACTCAGAGC39                                     (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      TTTAGTGGTACCTTTCTATTCTCACTCGGCCGAAACTGT39                                     (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AAAGCGCAGTCTCTGAATTTACCG24                                                    (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AlaGluXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa                              151015                                                                        XaaProProProProProPro                                                         20                                                                            __________________________________________________________________________

What is claimed:
 1. A method of screening for a test candidate thatbinds an endothelin B₁ (ETB₁) receptor polypeptide and modulates signaltransduction activity comprising:(a) providing a host cell transformedwith a DNA encoding endothelin B₁, receptor (ETB₁) polypeptide havingSEQ ID NO:1; (b) exposing said cell to said test candidate; and (c)measuring endothelin B₁ receptor signal transduction activity.
 2. Amethod of screening for a test candidate that binds an endothelin B₁(ETB₁) receptor polypeptide and modulates signal transduction activitycomprising:(a) providing a host cell transformed with a DNA encodingendothelin B, receptor (ETB₁) polypeptide having SEQ ID NO:1; (b)exposing said cell to said test candidate; (c) lysing said cell; and (d)measuring endothelin B₁ receptor signal transduction activity.